text:
Eclair allows you to scale up one _logical_ lightning node across multiple servers.

Front servers take care of routing table related gossip and syncing requests from peers, which is cpu/bandwidth intensive. The backend server can focus on core channel management. So, BOLT 1&7 messages are handled in the frontend, while BOLT 2 messages go through and are processed in the backend.

Front servers are stateless, they can be stopped/killed at will. The node will remain operational and reachable as long as there is at least one `frontend` available.

```
                +---+     +-----------+
                |   |     | +-------+ |
                |   |-----|-|       | |
       P   -----| L |-----|-| FRONT |-|---,
       U        | O |-----|-|       | |    \
       B     ---| A |     | +-------+ |     \
       L        | D |     |           |      \
       I        |   |     | +-------+ |       \ +------+
       C   -----| B |-----|-|       | |        `|      |
                | A |-----|-| FRONT |-|---------| BACK |<-- channels management
       N        | L |-----|-|       | |        ,|      |    relay logic
       E    ----| A |     | +-------+ |       / +------+
       T        | N |     |           |      /
       W        | C |     | +-------+ |     /
       O        | E |-----|-|       | |    /
       R   -----| R |-----|-| FRONT |-|---'
       K        |   |-----|-|       |<------- connection management
                |   |     | +-------+ |       routing table sync
                +---+     +-----------+
```

The goal is to offload your node from connection and routing table management:
- incoming connections
- outgoing connections
- gossip queries + pings
- incoming gossip aggregation
- outgoing gossip dispatch (rebroadcast)

### Prerequisite

You already have a lightning node up and running in a standalone setup (with Bitcoin Core properly configured, etc.).

You know what your `node id` is.

Conventions used in this document:
- what we previously called `eclair-node` will be called `backend`. It is to be launched, configured and backed-up exactly like in a standalone setup.
- `node` refer to *akka cluster nodes*, not to be confused with lightning nodes. Together, all *cluster nodes* form a single logical *lighting node*.

### Minimal/Demo setup

Use this if you want to experiment with the cluster mode on a single local server.

Set the following values in `.eclair/eclair.conf`:
```
akka.actor.provider = cluster
akka.extensions = ["akka.cluster.pubsub.DistributedPubSub"]

// replace this with your node id
// if you don't know what your node id is, you should probably stop right here
eclair.front.pub = 03...............
```

Start the `backend`:
```shell
$ ./eclair-node.sh
```

Then run an instance of `frontend`:
```shell
$ ./eclair-front.sh -Dakka.remote.artery.canonical.port=25521 -Declair.server.port=9736
```

NB: we override the ports, otherwise they would conflict since in this example everything runs on the same server. You can run multiple `frontend`s on the same server, just make sure to change the ports.

### Production setup

In production you should:
- run multiple `frontend` servers
- run one app per server
- enable `tcp-tls` to encrypt communications between members of the cluster with your own generated certificate (see below)
- use a load balancer to hide all your `frontend` servers under the same ip address
- set firewall rules to disable lightning connections (port 9735) on your `backend` server, so all connections go through the `frontend`
- enable [monitoring](Monitoring.md)
- on AWS, use AWS Secrets Manager (see [AWS deployment](#aws-deployment))

#### Enable encrypted communication for the cluster

We use a self-signed certificate, which offers a good compromise. More advanced options are available, see [akka doc](https://doc.akka.io/docs/akka/current/remoting-artery.html#remote-security).
> Have a single set of keys and a single certificate for all nodes and disable hostname checking
> - The single set of keys and the single certificate is distributed to all nodes. The certificate can be self-signed as it is distributed both as a certificate for authentication but also as the trusted certificate.
> - If the keys/certificate are lost, someone else can connect to your cluster.
> - Adding nodes to the cluster is simple as the key material can be deployed / distributed to the new cluster node.

Generate a self-signed certificate (set a strong password):
```shell
$ keytool -genkeypair -v \
          -keystore akka-cluster-tls.jks \
          -dname "O=ACME, C=FR" \
          -keypass <password> \
          -storepass <password> \
          -keyalg RSA \
          -keysize 4096 \
          -validity 9999
```

Copy the resulting certificate to the `.eclair` directory on your backend node and all your frontend nodes:
```shell
$ cp akka-cluster-tls.jks ~/.eclair
```
Add this to `eclair.conf` on all your frontend nodes:
```
akka.remote.artery.transport = "tls-tcp"
```

#### Run cluster nodes on separate servers

Start all your frontend nodes with the following environment variables:
* BACKEND_IP set to the IP address of your backend node
* LOCAL_IP set to the IP address of this frontend node (this is typically a private IP address, reachable from your backend node)
* NODE_PUB_KEY set to your node public key
* AKKA_TLS_PASSWORD set to the password of your Akka certificate

Add this to `eclair.conf` on your backend node:
```
akka.remote.artery.transport = "tls-tcp"
akka.remote.artery.canonical.hostname="ip-of-this-backend-node"
akka.cluster.seed-nodes=["akka://eclair-node@ip-of-this-backend-node:25520"]
```

Start your backend node with the following environment variables:
* AKKA_TLS_PASSWORD set to the password of your Akka certificate

### AWS Deployment

For convenience, we provide a prebuilt AWS Beanstalk bundle for the `frontend` (choose a WebServer environment type, and Java platform).

You can run it as-is for testing.

#### TLS encryption

If you intend to use it in production, you need to enable encryption with your own certificate:
1. Follow the procedure above to generate your `akka-tls.jks`

2. We recommend forking the project and building your own bundle:
```shell
$ git clone git@github.com:ACINQ/eclair.git
$ vi eclair-core/src/main/reference.conf # set akka.remote.artery.transport = "tls-tcp"
$ cp akka-cluster-tls.jks eclair-front/modules/awseb/ # copy the file you generated
$ vi eclair-front/modules/awseb.xml # uncomment the relevant parts
$ mvn package -DskipTests
```
Alternatively, you can also edit the existing bundle and manually add the `akka-cluster-tls.jks` file to the root of the zip archive. You will also need to set `akka.remote.artery.transport=tls-tcp` at runtime.

#### Private key

In production, we highly recommend using AWS Secrets manager to provide the node private key. This is done by setting `eclair.front.priv-key-provider=aws-sm`. Default secret name is "node-priv-key", but it is configurable with `eclair.front.aws-sm.priv-key-name`

#### Configuration

We recommend using Beanstalk environment variables for `AKKA_TLS_PASSWORD`, `BACKEND_IP`, and `NODE_PUB_KEY`. Other configuration keys should be set in the `AKKA_CONF` environment variable, semicolon separated. Example:
- `AKKA_CONF`: `eclair.enable-kamon=true; akka.remote.artery.transport=tls-tcp; eclair.front.priv-key-provider=aws-sm...`
- `AKKA_TLS_PASSWORD`: `xxxxxxxx`
- `BACKEND_IP`: `1.2.3.4`
- `NODE_PUB_KEY`: `03933884aaf1d6b108397e5efe5c86bcf2d8ca8d2f700eda99db9214fc2712b134`

Port `25520` needs to be open, within the Beanstalk security group, and between the Beanstalk security group and your `backend` node.

### Tor

We recommend running your Tor hidden service on a separate server, and use `eclair.tor.targets` to redirect clearnet(*) connections to your `frontend` servers.

(*) Clearnet for Tor, but BOLT 8 encrypted.

Here is the resulting architecture:

```
                             +---+     +-----------+
                             |   |     | +-------+ |
                             |   |-----|-|       | |
       P                -----| L |-----|-| FRONT |-|---,
       U                     | O |-----|-|       | |    \
       B                  ---| A |     | +-------+ |     \
       L                     | D |     |           |      \
       I                     |   |     | +-------+ |       \ +------+
       C              -------| B |-----|-|       | |        `|      |
                             | A |-----|-| FRONT |-|---------| BACK |<-- channels management
       N                     | L |-----|-|       | |        ,|      |    relay logic
       E                 ----| A |     | +-------+ |       / +------+
       T                     | N |     |           |      /
       W      +-------+      | C |     | +-------+ |     /
       O      |       |      | E |-----|-|       | |    /
       R   ---|  Tor  |------| R |-----|-| FRONT |-|---'
       K      |       |      |   |-----|-|       |<------- connection management
              +-------+      |   |     | +-------+ |       routing table sync
                             +---+     +-----------+
```

redpajama_set_name: RedPajamaGithub


text:
Q: Pull SKU, Manufacturer and custom attribute from Magento database I'm trying to pull a full list of products from the database with the SKU, manufacturer and a custom attribute called 'GTIN'.
I'm really struggling with the custom attribute part.
This statement works to pull the manufacturer and SKU
SELECT cpe.sku, m1.manufacturer,
FROM catalog_product_entity AS cpe
INNER JOIN exportview_manufacturer AS m1 ON cpe.entity_id = m1.entity_id

My MySQL is very poor and I can't seem to get this custom attribute. I've found the following statement online which I KNOW has all the details I need to make this work but thus far I've been getting my head in a mess trying to implement it
SELECT e.entity_id AS product_id, var.value AS product_name
FROM catalog_product_entity e, eav_attribute eav, catalog_product_entity_varchar var
WHERE
e.entity_type_id = eav.entity_type_id
AND eav.attribute_code = 'gtin'
AND eav.attribute_id = var.attribute_id
AND var.entity_id = e.entity_id

I simply need the first statement to also include the 'gtin' column, but joining is where I'm falling short. Can someone please assist?

A: You need to create one PHP script to get all product and it's attribute. So first create all_product.php file on your Magento root and add below code.
error_reporting(E_ALL);
ini_set("display_errors", 1);

require_once '../app/Mage.php';
Mage::app();

 $collection = Mage::getModel('catalog/product')->getCollection()
->addAttributeToSelect('*') // select all attributes
->setPageSize(5000) // limit number of results returned
->setCurPage(1); // set the offset (useful for pagination)

foreach ($collection as $product) {
 echo $product->getName(); //get name
/*you can get any attribue you want like name above. Even you can get all custom attribule for product for that you need to use print_r($collection->getData()) above foreach*/
}

Hope it will work.

redpajama_set_name: RedPajamaStackExchange


text:
Opinion: Making Congress smart again
By Wayne T. Brough and Josh WithrowInsideSources.com
Artificial intelligence, 5G, BitCoin and the Internet of Things. Rapid advances in technology are everywhere, and Congress is increasingly being asked to opine on all of it. Which is terrifying when you consider that the same senators and representatives who will be voting on the laws that guide the next generation of innovation are asking Google's CEO why their iPhone won't work and don't seem to understand Facebook is funded by advertising.
However, an increasingly bipartisan self-awareness of Congress' deficiencies in science and technology led to the creation of the office of Science, Technology Assessment and Analysis (STAA) inside the Government Accountability Office. This is an expansion of a small but successful pilot program started back in 2002, which in turn was an attempt to replace Congress' original scientific advisers, the Office of Technology Assessment (OTA).
Without this expertise, the void in information will be filled by others, either in the executive branch agencies implementing legislative mandates, or special interests pursuing their own agendas. Providing members of Congress with assessments of science and technology issues reduces information asymmetries between the branches of government. Additionally, technology assessments provided to members of Congress can enrich the congressional record and leave implementing agencies with far less discretion when it comes to interpreting legislative mandates. All of which can be a useful check on the growing administrative state.
In addition to participating in the legislative process, however, the GAO's main mission as an oversight and auditing agency gives it the statutory tools to pry information from executive branch agencies. Regulations from agencies as diverse as the FCC, the FDA and the SEC have a major impact on emerging technologies. As Congress attempts to oversee the bureaucracy its laws have created, having a team that combines the GAO's institutional knowledge of inspecting executive agencies with the expertise to report back on the technological impact of their regulations could function as an important check on executive branch overreach. In fact, such a capability will probably be necessary should Congress choose to reassert itself as the dominant policymaking branch of government.
The STAA's process for producing technology analyses will have to be structured to carefully avoid some of the pitfalls that befell the old OTA. Most important, they will have to take care to build a structure and culture that preserves its nonpartisan neutrality so that it can resist the wrath of one party or the other for merely producing unwelcome information. The OTA was dogged by accusations of partisan bias by Republicans (sometimes fairly, particularly in the early years), who were in the minority for most of its existence and promptly abolished the office within the first year of their new majority in 1995.
Not being bound by the OTA's governing structure will be one key reason the STAA can avoid its predecessor's fate. The OTA's research queue was approved by an advisory board that had equal representation from both major parties and each chamber, but whose members were picked by the majority party's leadership.
Partisanship in Congress has not exactly decreased in the quarter-century since the OTA, making the GAO's new program a more politically feasible option. Further, the GAO's request procedures emphasize committee and subcommittee inquiries, and should provide for a wider range of voices in Congress to avail themselves of its services.
In short, the new STAA program has the potential to exceed the program that it was designed to replace, by eliminating some structural concerns and focusing more directly on the specific needs of Congress. Toward this end, the GAO recently released a plan highlighting the STAA program and its ability to provide technology assessments on a wide range of issues, from the Internet of Things, to artificial intelligence, to cybersecurity. Having such technical expertise available on a real-time basis that is not beholden either to the executive branch or to lobbying interests can greatly aid in both legislation and regulatory oversight that reflects how relevant technologies actually work.
As Congress considers appropriations for the legislative branch, it must determine whether the STAA is in accord with its actual needs, and to provide any legal framework necessary to allow the STAA to fully achieve that mission. In so doing, it's important that nostalgia for the old OTA — which remains strong in some corners, not win out over the opportunity to develop of a program that may be even better suited to help make Congress smart again.
Wayne T. Brough is president of the Innovation Defense Foundation and Josh Withrow is a research fellow with the foundation. They wrote this for InsideSources.com.

redpajama_set_name: RedPajamaCommonCrawl


text:
Power Interest And Identity In Military Alliances PDF EPUB Download
Power Interest And Identity In Military Alliances also available in docx and mobi. Read Power Interest And Identity In Military Alliances online, read in mobile or Kindle.
Power, Interest, and Identity in Military Alliances
Author: J. Suh
Category: Social Science
This book looks at U.S.-Korea relations and argues that military alliances depend upon a combination of power distribution, material assets, and identities. The author asserts that beyond being mere tools of power balancing, alliances are also impacted by material and institutional practices that constitute the identity of allies and adversaries.
Author: J. J. Suh
Publisher: Macmillan
This book looks at U.S.-Korea relations and argues that the durability of military alliances depends upon a combination of power distribution, material assets, and identities. The author asserts that military alliances, beyond being mere tools of power balancing, are also engaged in material, representational, and institutional practices that constitute the identity of allies and adversaries.
Shifting Power in Asia-Pacific?
The Rise of China, Sino-US Competition and Regional Middle Power Allegiance
Author: Enrico Fels
This book investigates whether a power shift has taken place in the Asia-Pacific region since the end of the Cold War. By systematically examining the development of power dynamics in Asia-Pacific, it challenges the notion that a wealthier and militarily more powerful China is automatically turning the regional tides in its favour. With a special emphasis on Sino-US competition, the book explores the alleged linkage between the regional distribution of relevant material and immaterial capabilities, national power and the much-cited regional power shift. The book presents a novel concept for measuring power in international relations by outlining a composite index on aggregated power (CIAP) that includes 55 variables for 44 regional countries and covers a period of twenty years. Moreover, it develops a middle power theory that outlines the significance of middle powers in times of major power shifts. By addressing political, military and economic cooperation via a structured-focused comparison and by applying a comparative-historical analysis, the book analyses in depth the bilateral relations of six regional middle powers to Washington and Beijing.
Truth and Reconciliation in South Korea
Between the Present and Future of the Korean Wars
Author: Jae-Jung Suh
The Korean War is multiple wars. Not only is it a war that began on 25 June 1950, but it is also a conflict that is rooted in Korea's colonial experiences, postcolonial desires and frustrations, and interventions and partitions imposed by outside forces. In South Korea, the war is a site of contestation: Which war should be remembered and how should it be remembered? The site has been overwhelmed by the Manichean official discourse that pits evil communists against innocent Koreans, but the hegemonic project remains unfinished in the face of the resiliency embodied in the survivors who have withstood multiple killings by the state. The historical significance of the Truth and Reconciliation Commission, Republic of Korea (TRCK), lies in its success in bringing back to life the voices of the silenced that complicate the hegemonic memory of the war as yugio, the "June 25th war." At the same time, the Commission embodies the structural dilemma that the effort to give voice to the silenced has turned to the state to redress the state's wrongdoings. The TRCK as such stands on the problematic boundary between violence and post-violence, insecurity and security, exception and normalcy. Truth and reconciliation, and human security, are perhaps located in a process of defining and redefining the boundary. This edited volume explores such political struggles for the future reflected in the TRCK's work on the past war that is still present. This book was published as a special issue of Critical Asian Studies.
Inside the Red Box
North Korea's Post-totalitarian Politics
Author: Patrick McEachern
Publisher: Columbia University Press
North Korea's institutional politics defy traditional political models, making the country's actions seem surprising or confusing when, in fact, they often conform to the regime's own logic. Drawing on recent materials, such as North Korean speeches, commentaries, and articles, Patrick McEachern, a specialist on North Korean affairs, reveals how the state's political institutions debate policy and inform and execute strategic-level decisions. Many scholars dismiss Kim Jong-Il's regime as a "one-man dictatorship," calling him the "last totalitarian leader," but McEachern identifies three major institutions that help maintain regime continuity: the cabinet, the military, and the party. These groups hold different institutional policy platforms and debate high-level policy options both before and after Kim and his senior leadership make their final call. This method of rule may challenge expectations, but North Korea does not follow a classically totalitarian, personalistic, or corporatist model. Rather than being monolithic, McEachern argues, the regime, emerging from the crises of the 1990s, rules differently today than it did under Kim's father, Kim Il Sung. The son is less powerful and pits institutions against one another in a strategy of divide and rule. His leadership is fundamentally different: it is "post-totalitarian." Authority may be centralized, but power remains diffuse. McEachern maps this process in great detail, supplying vital perspective on North Korea's reactive policy choices, which continue to bewilder the West.
Bound to Last
Power, Interest, and Identity in the U.S.-Korea Alliance
Author: Chae-jŏng Sŏ
Category: Alliances
Category: Academic libraries
Peace, Power, and Order in East Asia
Author: David C. Kang
Throughout the past three decades East Asia has seen more peace and stability than at any time since the Opium Wars of 1839-1841. During this period China has rapidly emerged as a major regional power, averaging over nine percent economic growth per year since the introduction of its market reforms in 1978. Foreign businesses have flocked to invest in China, and Chinese exports have begun to flood the world. China is modernizing its military, has joined numerous regional and international institutions, and plays an increasingly visible role in international politics. In response to this growth, other states in East Asia have moved to strengthen their military, economic, and diplomatic relations with China. But why have these countries accommodated rather than balanced China's rise? David C. Kang believes certain preferences and beliefs are responsible for maintaining stability in East Asia. Kang's research shows how East Asian states have grown closer to China, with little evidence that the region is rupturing. Rising powers present opportunities as well as threats, and the economic benefits and military threat China poses for its regional neighbors are both potentially huge; however, East Asian states see substantially more advantage than danger in China's rise, making the region more stable, not less. Furthermore, although East Asian states do not unequivocally welcome China in all areas, they are willing to defer judgment regarding what China wants and what its role in East Asia will become. They believe that a strong China stabilizes East Asia, while a weak China tempts other states to try to control the region. Many scholars downplay the role of ideas and suggest that a rising China will be a destabilizing force in the region, but Kang's provocative argument reveals the flaws in contemporary views of China and the international relations of East Asia and offers a new understanding of the importance of sound U.S. policy in the region.
Studies in Contemporary Jewry: XI: Values, Interests, and Identity
Jews and Politics in a Changing World
Author: Avraham Harman Institute of Contemporary Jewry
Publisher: OUP USA
This collection of original articles addresses the often conflicting roles of values, interests, and identity in contemporary Jewish politics. with its focus on Jews and contemporary politics - particularly the interplay of politics and jewish history - this new work makes an outstanding contribution to the scholarly literature.
Nationalism in Europe and America
Politics, Cultures, and Identities since 1775
Author: Lloyd S. Kramer
Publisher: UNC Press Books
Examining the history of nationalism's pervasive influence on modern politics and cultural identities, Lloyd Kramer discusses how nationalist ideas gained emotional and cultural power after the revolutionary upheavals in the late eighteenth-century Atlantic world. Nationalism in Europe and America analyzes the multiple historical contexts and intellectual themes that have shaped modern nationalist cultures, including the political claims for national sovereignty, the emergence of nationalist narratives in historical writing and literature, the fusion of nationalism and religion, and the overlapping conceptions of gender, families, race, and national identities. Kramer emphasizes the similarities in American and European nationalist thought, showing how European ideas about land, history, and national destiny flourished in the United States while American ideas about national independence and political rights reappeared among European nationalists and also influenced the rise of anticolonial nationalisms in twentieth-century Asia and Africa. By placing nationalist ideas and conflicts within the specific, cross-cultural framework of Atlantic history and extending his analysis to the twentieth-century world wars, Kramer offers readers a thoughtful perspective on nationalism's enduring political and cultural importance throughout the modern world.
If My Heart Had Wings
Ketogenic Diet Cookbook
Saffron Lane
Build Your Own Lego Vehicles
Reflections on the Unknowable
Letters to Open When...
On Writing History from Herodotus to Herodian
The Secret of Happy Children
Econometric Analysis: International Edition
Poetry of Place
Write It Up
The Winter of the Witch
Scaling Lean
Photojournalism: The Professionals' Approach
Global Shift
A Christmas Treasury
Handbook of Critical and Indigenous Methodologies
The Colour Illustrated Hobbit
And Now The Shipping Forecast
Permission to Mourn
Calculated in Death
BMW R850, 1100 & 1150
Whispers from God
The New Cosmic Onion
What the hell happened to my brain?
Phonics from A to Z
Chasing the Night (Eve Duncan Book 11)
Rad des Tijds / 12 De naderende storm / druk 1
The Corruption of Capitalism
Oil & Acrylic: Land and Sea
Rhythms, Rites and Rituals
Mona Lisa Awakening
Environmental Land Use Planning and Management
Fountas & Pinnell Genre Study
Indian Superfood
Narrative Counseling in Schools
The Workbench Guide to Jewelry Techniques
Second Term at Trebizon
Starting Endurance Riding
Key element guide ITIL service transition
The Crash Course
Early Reader: Pretty Poodle Parlour
The World Beyond Your Head
Mi prima Carolina/ Red Eyes at Night
The Colour Thief
Torchon Lacemaking
Defeat Into Victory
Secrets in the Fire
Every Woman's Guide to Foot Pain Relief
A Year In The Merde
Pelagia and the White Bulldog
Complete Book of Throws
Stolen Songbird
Wage-Labour and Capital & Value, Price and Profit
Paddy Whacked
Here I Am: A Novel
SmallTalk, Objects, and Design
Ecology of Wisdom
El secreto / The Secret History


Have you polished your presentation skills? Or, are you like many agents–just going to the door and starting 'naturally'. We're all presenters: Any time we're in front of two or two thousand, our goal is to persuade the audience to our point of view. However, most of the time, we just get in front of people and say whatever we think of first. That lack of attention to presentation organization leads to some big presentation mistakes, and costs us 'sales'. Instead of stumbling through a presentation, why not organize it to grab their attention, persuade them to your way of thinking, and motivate them to action?
Many more tips on presentations and presentation skills are in my new resource, Knock Their Socks Off: Tips to Make your Best Presentation Ever.
Do You Have a Listing Presentation or a Marketing Plan?


Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons) can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (sh)RNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB), discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.
DNA transposons are discrete pieces of DNA with the ability to change their positions within the genome via a 'cut and paste' mechanism called transposition. In nature, these elements exist as single units containing the transposase gene flanked by terminal inverted repeats (TIRs) that carry transposase binding sites (Figure 1A). However, under laboratory conditions, it is possible to use transposons as bi-component systems, in which virtually any DNA sequence of interest can be placed between the transposon TIRs and mobilized by trans-supplementing the transposase in the form of an expression plasmid (Figure 1B) or mRNA synthesized in vitro. In the transposition process, the transposase enzyme mediates the excision of the element from its donor plasmid, followed by reintegration of the transposon into a chromosomal locus (Figure 1C). This feature makes transposons natural and easily controllable DNA delivery vehicles that can be used as tools for versatile applications, ranging from somatic and germline transgenesis to functional genomics and gene therapy (Figure 2).
General organization and use of class II transposable elements as gene vectors. (A) Autonomous transposable elements consist of terminal inverted repeats (TIR; black arrows) that flank the transposase gene. (B) Bi-component transposon vector system for delivering transgenes that are maintained in plasmids. One component contains a DNA of interest between the transposon TIRs carried by a plasmid vector, whereas the other component is a transposase expression plasmid, in which the black arrow represents the promoter driving expression of the transposase. (C) The transposon carrying a DNA of interest is excised from the donor plasmid and is integrated at a chromosomal site by the transposase.
Broad applicability of transposon-based gene vectors in vertebrate genetics.
Transposons have been successfully used in plants and in invertebrate animal models, including Arabidopsis, rice, Caenorhabditis elegans[1–3] and Drosophila[4–6] for transgenesis and insertional mutagenesis, but until recently, there was no known transposon that was sufficiently active to be tailored as a tool for such purposes in vertebrates. This is because transposons tend to have limitations with respect to the species in which they can jump. In 1997, the Sleeping Beauty (SB) transposon system was engineered by molecular reconstruction of an ancient, inactive Tc1/mariner-type transposon found in several fish genomes . This newly reactivated element allowed highly efficient transposition-mediated gene transfer in major vertebrate model species without the potential risk of cross-mobilization of endogenous transposon copies in host genomes. This is because the genomes of major models lack endogenous transposon sequences with sufficient sequence similarity for mobilization by an exogenously supplied SB transposase. Indeed, SB has been successfully used as a tool for genetic modifications of a wide variety of vertebrate cell lines and species including humans [8–10].
During the past decade, other elements have been shown to catalyze efficient transposition in vertebrate model organisms. For example, the insect elements piggyBac[11, 12] and Minos[13, 14] catalyze efficient transposition in mammalian cells. Minos was also shown to be active in the basal chordate Ciona intestinalis. Moreover, the reconstructed amphibian element Frog Prince, the reconstructed human Hsmar1 element , the reconstructed zebrafish transposon Harbinger3_DR, and the Tol1 and Tol2 elements isolated from the medaka fish have been found to be active in vertebrate species. Passport, a Tc1-family transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells , and the Ac/Ds transposon originally discovered in maize by McClintock undergoes efficient transposition in zebrafish embryos . Thus, the piggyBac, Minos and Ac/Ds elements appear to have a significantly wider possible host range than most other transposons. The basic criteria for the applicability of a transposon in any given model organism are 1) a sufficient level of transpositional activity in the given species, and 2) target site selection properties of the transposon, which are discussed below.
In evolutionary terms, the SB transposon represents a successful element that was able to colonize several fish genomes millions of years ago . However, even successful transposons have not been selected for the highest possible activity in nature, because unlike viruses, they have to coexist with their hosts and, consequently, there is strong selective pressure to avoid insertional mutagenesis of essential genes. Indeed, although the resurrected SB element was sufficiently active to be mobilized in vertebrate cells, its relatively limited transpositional activity still presented a bottleneck for some applications. For example, requirements for transfection of primary cells and other hard-to-transfect cell types, or for remobilization of transposons from chromosomally resident single-copy donor sites, demanded an enzyme with more robust activity. Thus, enhancing transpositional activity has been one of the main targets for transposon vector development. To date, almost every single amino acid in the SB transposase has been changed in an attempt to increase its activity. Three main strategies have been applied to derive hyperactive mutants of the SB transposase: 'importing' amino acids and small blocks of amino acids from related transposases [23–25], systematic alanine scanning , and rational replacement of selected amino acid residues . Together, these studies have yielded several, single amino acid replacements, each resulting in a relatively modest increase in transpositional activities. Unfortunately, the hyperactivity of most of the SB transposase mutants selected in immortalized cell lines did not translate to efficient stable gene transfer in primary cells in vivo[25, 26].
Earlier studies established that certain combinations of amino acid replacements, each leading to hyperactivity, can yield a further enhancement in transpositional activity of the SB transposase [23–26], but guessing the correct combinations of variants out of the millions that are possible is like finding the correct combinations of numbers in a lottery. A high-throughput, PCR-based, DNA-shuffling strategy and screening of 2000 gene variants in mammalian cells produced a variant of SB that was 100-fold more potent in chromosomal insertion of a transgene than the originally reconstructed protein . The use of SB100X demonstrated that it is possible to establish a transposon-based, non-viral vector system that is capable of stable gene transfer coupled with long-term gene expression at an efficiency comparable with that of viral strategies . Thus, the hyperactive SB100X transposase holds great promise of offering broad utility in gene therapy and functional genomics.
The insertion pattern of most transposons is nonrandom, showing characteristic preferences for insertion sites at the primary DNA sequence level, and 'hotspots' and 'cold regions' on a genome-wide scale. For example, for the primary DNA sequence, the Tol2 element does not appear to exhibit a pronounced preference for any sequence for insertion . By contrast, the Harbinger3_DR transposon is highly specialized to integrate into the palindromic AAACACCWGGTCTTT consensus sequence , the piggyBac transposon targets the sequence TTAA, and all Tc1/mariner transposons, including SB, Frog Prince, Minos and Hsmar1, target their integration into TA dinucleotides. In the case of SB, this preference has been studied in detail, and palindromic AT repeats found to be the preferred sites for integration . However, computational analyses revealed that target selection is determined primarily at the DNA structure level, not by specific base-pair interactions. For example, protein-induced deformability was shown to be associated with preferred SB insertion sites, whereas piggyBac and Tol2 integration sites lack such consistent, clear-cut structural patterns [30, 31]. This suggested that integrations of SB will occur into any DNA available, depending on these preferences only, but this is not the case. In the context of chromatin, Tc1/mariner elements have no or weak preference for transcription units, the 5' regulatory regions are not favored, and most hits in genes are localized within introns [29, 32]. By contrast, piggyBac shows a greater propensity to integrate into transcription units, with a preference for insertion around transcription start sites [12, 33–35], and the Tol2 transposon also shows a pronounced preference for integration close to transcriptional start sites . This control of integration at the chromatin level is poorly understood. One possible explanation for this is the interaction of the transposase with unknown, chromatin-associated factors. Supporting this hypothesis, it has been shown that a host-encoded protein, lens epithelium derived growth factor (LEDGF), is involved in directing integration of human immunodeficiency virus (HIV) into active genes . Taken together, the preferences of particular elements to integrate into expressed genes versus non-coding DNA, and their preferences for integration sites within genes are expected to be substantially different.
Integration site preference can greatly influence the utility of transposon vectors for different applications. For example, human gene therapy protocols require application of transposon vectors showing the least preference for target genes, for obvious safety reasons. The SB system (which shows close-to-random insertion site distribution) appears to best satisfy these needs, whereas the piggyBac and Tol2 systems (which prefer genes and their upstream regulatory regions for insertion) appear to be less favorable for potential therapeutic applications. Nevertheless, a systematic assessment of potential genotoxic effects associated with genomic integration of transposon vectors will need to be performed either in cell-based assays and/or in animal models to provide clinically relevant data.
Unlike in therapeutic applications, hitting genes by insertional elements is the goal with forward mutagenesis screens. However, the insertional biases associated with vector systems represent the main limitation to full genome coverage with individual transposon-based vectors. Thus, in this respect, the utility of transposons for mutagenesis is greatly enhanced by the availability of multiple alternative vector systems with distinct preferences for insertion, such as SB, Tol2 and piggyBac. Indeed, the propensity of Tol2 to insert close to transcriptional start sites of genes might be particularly advantageous for enhancer trapping [37, 38], while the propensity of piggyBac to insert into transcription units supports genome-wide mutagenesis with gene trap cassettes .
'Local hopping' describes a phenomenon of chromosomal transposition in which transposons have a preference for landing into cis-linked sites in the vicinity of the donor locus. Local hopping seems to be a shared feature of 'cut and paste' transposons. However, the actual extent of hopping to linked chromosomal sites and the interval of local transposition varies. For example, the P-element transposon of Drosophila prefers to insert within ~100 kb of the donor site at a rate ~50-fold higher than in regions outside that interval . Similarly, in germline mutagenesis screens in mice using SB, 30-80% of the transposons re-insert locally on either side of the transposon donor locus [41–43]. In contrast to the P-element, SB seems to have a much larger local transposition interval between 5 and 15 Mb .
The local hopping feature not only differs between different transposons, but a given transposon may show great variations in local hops in different hosts, and in different donor loci even in the same host. For example, about 50-60% of the reinserted Ac elements were found to be distributed within a 5-cM distance of the donor site in maize [44, 45], and the frequency of local hopping greatly varies in Arabidopsis and tobacco, depending on the chromosomal location of the donor site [46–48]. Moreover, local hopping of the Ac element in tomato seems overall to be less prevalent than in maize [49, 50], and there are species-specific differences in its tendency for local hopping out of different transposon donor loci . This variation in local hopping of the same element could possibly be explained by varying affinity of the transposase to unknown, chromatin-associated factors in different hosts .
Local hopping can play a significant role in mutagenesis using chromosomally resident transposons. In practical terms, local hopping limits the chromosomal regions accessible to a transposon jumping out of a given chromosomal site . To circumvent this limitation, establishing numerous 'launch pads' to initiate transposition out of different loci can be a viable strategy to increase coverage of gene mutations. On the other hand, local hopping can be useful for saturation mutagenesis within limited chromosomal regions.
The post-genomic era presented the scientific community with the new challenge of functional annotation of every gene and identification of elaborate genetic networks. Diverse methods have been employed to address this task, including mutational analysis, which proved to be one of the most direct ways to decipher gene functions. There are versatile strategies for creating mutations, including insertional mutagenesis by discrete pieces of foreign DNA, which has the advantage that the inserted DNA fragment can serve as a molecular tag that allows rapid, usually PCR-based, identification of the mutated allele. Because the function of the gene in which the insertion has occurred is often disturbed, such loss-of-function insertional mutagenesis is frequently followed by functional analysis of mutant phenotypes. In many instances, retroviral vectors were used to introduce mutagenic cassettes into genomes, but their chromosomal insertion bias does not allow full coverage of genes . The random integration pattern of the SB transposon, combined with its ability to efficiently integrate versatile transgene cassettes into chromosomes established this system as an extremely useful tool for insertional mutagenesis in both embryonic stem cells (ESCs) [34, 55] and in somatic [56, 57] and germline tissues [41, 42, 53, 58–63] in animal models (Figure 2). There are several types of mutagenic cassettes that can be efficiently combined with transposon-based gene delivery for insertional mutagenesis. 5' gene-trap cassettes include splice acceptors and polyadenylation sequences so that transcription of genes can be disrupted upon vector insertion into introns (Figure 3A) . Often, such cassettes are also equipped with a reporter gene (usually, a fluorescent protein, β-galactosidase or antibiotic resistance) whose expression is dependent on correct splicing between exons of the trapped gene and the splice acceptor site carried by the transposon vector [64, 65].
Major types of expression and mutagenic cassettes delivered by transposon vectors for versatile applications. (A) A gene-trap transposon contains a splice acceptor (SA) sequence followed by a promoterless reporter gene such as lacZ and a poly-A (pA) signal. The reporter gene is expressed only when its transcription is initiated from the promoter of a disrupted, actively transcribed endogenous transcription unit. Therefore, the expression pattern of the transposon reporter reflects that of the endogenous gene harboring the transposon insertion. (B) Oncogene-trap transposons include a strong viral promoter/enhancer long terminal repeat (LTR) sequence that can drive transcription toward the outside of the transposon and a splice donor (SD) sequence, so that forced transcription and splicing to downstream exons will result in overexpression of a product of a gene into which the transposon has inserted. Classic oncogene trap vectors also contain SA and pA sites to induce gene truncations that have dominant phenotypes. (C) Knockdown expression cassette including a polymerase (Pol) II promoter that drives expression of a marker gene and a Pol III promoter that drives expression of a short hairpin (sh)RNA. (D) A typical therapeutic expression cassette contains a ubiquitous or tissue-specific enhancer/Pol II promoter that drives expression of a therapeutic gene. To enhance the safety of such a vector, the expression cassette might be flanked by insulator elements that will block transactivation of endogenous promoters by the transposon insertion, and simultaneously protect the expression of the therapeutic gene from position effects.
Insertional mutagenesis can be applied to cultured, germline-competent stem cells including ESCs and spermatogonial stem cells (SSCs) [39, 66]. One advantage of this approach is the ability to perform preselection of modified ESC clones before generating mutant animals, and to differentiate selected clones into many different tissue types in vitro. It is possible to perform large-scale, transposon-based, insertional mutagenesis screens in ESCs and SSCs by simply transfecting or electroporating transposon donor and transposase expression plasmids into the cells. The amounts of the delivered plasmids can be adjusted to obtain the desired insertion frequencies per cell. In addition, transposons can also be remobilized from chromosomally resident loci and reintegrated somewhere else in the genome by transiently providing the transposase source; such excision-re-integration events can be monitored using double selection systems, in which excision activates the first and re-integration activates the second selection marker .
Because several aspects of physiology in rats have evolved to be more similar to humans than to mice, it would be desirable to use rat models in the process of functionally annotating the human genome by identifying the causative relationships between genes and disease phenotypes. As an important step towards this goal, an approach of establishing SB transposon-mediated insertional mutagenesis in rat SSCs was recently reported . SB transposition can be used to tag and simultaneously mutate thousands of genes in culture, using gene-trap cassettes. Importantly, culture conditions maintain the potential of genetically manipulated SSCs to produce viable sperm cells. In that study, spermatogonial clones were transplanted to repopulate the testes of sterilized, wild-type recipient male rats. The stem cell genome was then passed on to transgenic offspring upon crossing of the recipient males with wild-type females (Figure 4). Although transposition events in a given target gene occur by chance, the tissue culture conditions allow screening for a large number of events. Transposition-mediated gene insertion and cell culture conditions thus allow generation of libraries of gene knockouts in rat SSCs (Figure 4). This technology has the potential to develop powerful genomic tools for use in the rat, offering the opportunity to create a bridge between physiology and genomics.
Generation of knockout rats by insertional mutagenesis with gene trap transposons in spermatogonial stem cells. Cultured stem cells are transfected with gene trap transposon and transposase constructs that will lead to thousands of transposon insertions covering all chromosomes. Those cells in which insertions have occurred in expressed genes can be selected based on activation of the gene trap marker, and the insertion sites can be mapped. Cell clones or polyclonal insertion libraries can be transplanted into the testes of sterile males, in which the spermatogonial step cells will undergo spermatogenesis. These transplanted males are crossed with wild-type females to pass the insertions through the germline and generate transgenic/knockout animals.
Another method in which transposons are used for insertional mutagenesis in animal models employs a 'jump-starter and mutator' scheme [42, 58, 61]. In this arrangement, mutator transgenic lines carry SB transposon-based gene-trapping vectors in the form of multicopy concatemers, whereas a jump-starter line expresses the transposase preferentially in the male germline [41, 64]. Crossing of the two lines results in transposition in the germline of the F1 double-transgenic males, which are then repeatedly crossed with wild-type females to segregate the transposition events that occurred in their sperm cells to separate F2 animals. In the mouse system, a single sperm cell of an F1 male contains, on average, two transposon insertions , and up to 90% of the F2 progeny can carry transposon insertions . The applicability of this approach has been demonstrated by the identification of mouse genes with either ubiquitous or tissue-specific expression patterns [42, 64, 67, 68]. Recently, a similar system for SB insertional mutagenesis was also established in rats [62, 63].
One cautionary note of launching transposition out of transposon arrays is that recombination between newly transposed transposon copies and the donor concatemer could lead to unwanted genomic rearrangements, as observed by Geurts et al. . The most likely explanation for the rearrangements is that transposition out of a concatemer generates new transposase binding sites, linked either in cis (in the case of local hops) or in trans (in the case of transposition onto other chromosomes). However, because some transposon copies remain at the original donor locus, transposase can recombine chromosomal sequences that are located between the individual transposon units by hybrid element transposition (that is, the end of one transposon pairs with the opposite end of another transposon at a different location) , leading to deletions and translocations. Such chromosomal rearrangements are unlikely to occur if a single-copy donor is used. Thus, transposon systems sufficiently active for efficient transposition out of single-copy donors might eliminate the need for concatemeric donor sites in animal breeding schemes. Indeed, the Tol2 element was demonstrated to show transposition at reasonable efficiencies when launched from singly-copy donor sites in transgenic zebrafish . In this context, the newly developed SB100X hyperactive system might also prove useful in future genetic screens.
Forward genetic screens do not necessarily need to depend on the breeding scheme described above; in some cases, a reasonable throughput in generation of transposon insertion mutants can be achieved by introducing the mutagenic transposon into individual animals, such as in zebrafish. A gene trap mutagenesis screen was recently employed to uncover genetic determinants of nicotine response in zebrafish, through a behavioral genetic screening paradigm . Using standard transposase-mediated transgenesis protocols, Tol2-based mutagenic vectors were co-injected into early zebrafish embryos by to generate a pool of mosaic F0 founder animals, which then underwent two successive rounds of crossing to generate homozygous mutant animals. Segregation of mutant animals from wild-type siblings was carried out using fluorescent reporters built into the gene trap cassettes. After profiling nicotine response in mutant versus wild-type fish, two mutants were identified out of a total of 102 fish lines screened . This study emphasizes the utility of transposons for the discovery and functional annotation of genes relevant to human health in forward, phenotype-driven genetic screens in model species.
To induce gain-of-function mutations, transposon vectors can be equipped with oncogene trap cassettes that contain strong viral enhancers/promoters that can drive transcription outwards from the vector, thereby leading to overexpression of a full-length or truncated protein product of the trapped gene, as well as splice acceptor and polyA sites that lead to gene truncation with dominant phenotypes (Figure 3B) [9, 72]. SB vectors harboring oncogene traps have been successfully used in large-scale cancer gene discovery screens in experimental animals (Figure 5) [10, 73, 74]. In these studies, SB transposons were somatically mobilized from donor chromosomal concatemers, which contained either low (25)  or high (150-350)  numbers of the oncogene trap transposon. Dominant mutations in somatic tissues of double transgenic mice carrying a transposase source and the mutagenic transposons resulted in the generation of experimental tumors in cancer-predisposed  and wild-type  animals. In a follow-up study, Collier et al. demonstrated that a combination of low-copy oncogene trap lines with the SB11 transposase (an early-generation hyperactive SB variant) expressed from the Rosa26 locus could achieve whole-body transposon mobilization at rates sufficient to promote penetrant tumorogenesis without complications of embryonic lethality or genomic instability . Thus, this approach can be successfully employed not only to identify novel cancer genes, but also combinations of cancer genes that act together to transform a cell.
Transposon-mediated cancer gene discovery screen. Breeding of 'jumpstarter' and 'mutator' stocks induces transposition in the soma of double-transgenic animals ('oncomice'). In the case of tissue-specific screens, a third genotype containing a tissue-specific Cre allele has to be crossed in to the backgrounds. The crosses can be made either in wild-type or in specific cancer-predisposed genetic backgrounds. Transposition in somatic cells leads to random insertional mutations, and animals are monitored for tumor development. Transposon insertions are cloned from genomic DNA isolated from tumor samples, and are subsequently mapped and the mutagenized genes identified. Those genes repeatedly mutated in multiple, independent tumors are designated as common insertion sites (CIS). These candidate cancer genes are then functionally validated.
Current efforts are concentrating on customized, tissue-specific screens for cancer development studies. The strategies employed to achieve this goal focus on establishing mouse lines that either conditionally express the transposase from tissue-specific promoters, or rely on generation of Cre recombinase-inducible transposase alleles that can be used in conjunction with mice that express Cre in a tissue-specific manner [76–78]. For example, this approach was addressed by Dupuy and co-workers , who were able to experimentally modify the spectrum of tumors by creating a Cre-inducible SB transposase allele (RosaSBaseLsL). With this strategy, they managed to overcome the obstacle of high embryonic lethality associated with ubiquitous SB transposase expression in the presence of the pT2/Onc2 oncogene trap [76, 79], and to generate a model of germinal center B-cell lymphoma. They achieved this by activating SB transposase expression with an AidCre allele that drove Cre-mediated recombination in germinal center B-cells. In another approach, ubiquituous expression of the SB transposase was combined with the novel T2/Onc3 oncogene trap transposon vector. In that study, the MSCV (mouse stem cell virus) 5' long terminal repeat that was previously used to drive oncogene expression was replaced by the ubiquitously active CAGGS promoter, resulting in removal of the bias towards inducing mostly lymphomas and in reducing embryonic lethality. This strategy emphasizes that the change in the design of the mutagenic transposon (e.g. promoter choice) can have profound effects on the tumor types induced by transposition. Notably, this approach resulted in production of nearly 200 independent tumors of more than 20 types, and identification of novel, candidate cancer genes, suggesting that the combination of tissue-specific promoters and inducible transposase alleles could provide a fine mechanism of control in tumorogenesis studies.
The classic approaches to stable expression of foreign genes in vertebrates rely on physical methods of delivering gene constructs into cultured cells, such as transfection, electroporation and sonoporation, or microinjection into oocytes or fertilized eggs to generate germline transgenic animals. The main drawbacks of these approaches are the low rates of genomic integration and the unstable expression of the chromosomally integrated gene construct, which is believed to be associated with the phenomenon of concatemerization of the injected DNA before genomic integration . Another particular problem in transgenic animals is that founders that develop from the injected oocytes or eggs are predominantly mosaic for the transgene, because integration generally occurs relatively late during embryonic development. In principle, all of these drawbacks can be circumvented by transposition-mediated gene delivery, as it can increase the efficiency of chromosomal integration and facilitates single-copy insertion events. Single units of expression cassettes are presumably less prone to transgene silencing than are the concatemeric insertions created by classic methods.
Transposon-based technologies can be exploited for gene transfer in cultured cells and in primary cell types, including stem cells (Figure 2). For example, transposons can be harnessed to integrate plasmid-based shRNA expression cassettes into chromosomes to obtain stable knockdown cell lines by RNA interference (Figure 3C) . Such technologies have been evaluated as a potential approach to the therapy of acquired immunodeficiency syndrome by stable RNA interference with SB vectors knocking down the CCR5 and CXCR4 cell surface co-receptors that are required for viral entry as a first step to confer resistance to HIV . Both the SB and the piggyBac systems were shown to support efficient transposition in mouse [33, 43] and human [83, 84] ESCs. In a recent, elegant study, piggyBac-derived transgene vectors were introduced into human ESCs for the purpose of driving ESC differentiation toward a specific cell type . The vectors included loss-of-function shRNA expression cassettes that could simultaneously knock down the expression of pluripotency genes and of genes that contribute to endodermal and mesodermal differentiation, plus a gain-of-function construct expressing Sox1 to direct differentiation towards neuroectoderm. What makes such complex gene-transfer experiments possible is the observation that several transgene constructs maintained on separate transposon vectors can be delivered simultaneously in 'multiplex' transposition reactions, in which the different constructs are simply mixed together and cotransfected into cells .
The recent discovery of induced pluripotent stem cells (iPSCs) by the expression of four key genes (Oct4, Sox2, Klf4 and c-Myc) in differentiated somatic cells holds enormous promise for future regenerative medicine . Transposons are attractive vehicles for reversible production of iPSCs, because the excision step of the transposition reaction produced by transient re-expression of the transposase offers removal of the transgenes after completion of reprogramming, allowing subsequent differentiation of the iPSCs into various lineages in vitro. Transposition-mediated generation of mouse and human iPSCs, and removal of the reprogramming factors from the pluripotent cells have already been achieved by the piggyBac system . What makes piggyBac transposons especially attractive vectors for the production of transgene-free iPSCs is a special feature of these transposons: excision fully restores the sequence of the original wild-type locus , thereby allowing traceless removal of transgenes from the genome.
In vivo, co-injection of engineered transposons with transposase mRNA into fertilized oocytes can facilitate early integration events that potentiate successful transmission of the transgene through the germline to the next generation (Figure 2). This method has been employed to generate transgenic zebrafish with Tc3, Mos1, Tol2 and SB; transgenic Xenopus with SB and Tol2; and transgenic mice with SB[27, 95–97], piggyBac and Tol2. In this context, an important step towards transgenesis with bacterial artificial chromosomes (BACs) has been made by the delivery of a ~70-kb BAC construct into zebrafish and mouse embryos with Tol2. Thus, transposons can evidently be used to stably deliver large transgene constructs together with complex regulatory regions, without the complications of DNA rearrangements and silencing associated with classic methods.
Any transgene vector system should provide long-term expression of transgenes. Transgenes delivered by non-viral approaches often form long, repeated arrays (concatemers) that are targets for transcriptional silencing by heterochromatin formation. In addition, long-term expression of transgenes delivered by retroviruses has been shown to be compromised by transcriptional silencing . It was recently shown that the zinc finger protein ZFP809 bridges the integrated proviral DNA of the murine leukaemia virus and the tripartite motif-containing 28 transcriptional co-repressor in embryonic stem cells . Thus, sequence elements in the vector itself can predispose the cargo for silencing. The cut and paste mechanism of DNA transposition results in a single copy of the transgene per insertion locus, thus concatemer-induced gene silencing is unlikely to be an issue with transposition-mediated gene transfer. Indeed, Grabundzija et al. found that transposon insertions delivered by the SB, Tol2 and piggyBac systems only rarely (< 4% of all insertions) undergo silencing in HeLa cells . Furthermore, stable transgene expression observed in hundreds of independent insertions in this study suggests that these three transposon systems rarely target heterochromatic chromosomal regions for insertion, and that it is unlikely that certain sequence motifs in the transposon vectors are recognized by mediators of silencing in the cell. An additional factor that may provoke transgene silencing is the cargo DNA, particularly the type of promoter used to drive expression of the gene of interest. Indeed, it was previously shown that transgene constructs delivered into mouse cells using SB transposition can be subject to epigenetic regulation by CpG methylation and that a determinant of epigenetic modifications of the integrating transposon vector is the cargo transgene construct, with the promoter playing a major role . However, with careful promoter choice, several studies have established that SB-mediated transposition provides long-term expression in vivo. For example, stable transgene expression from SB vectors was seen in mice after gene delivery in the liver [103–106], lung [107, 108], brain  and blood after hematopoietic reconstitution in vivo[27, 110]. Thus, although our understanding of all the factors that will ultimately determine the expressional fate of an integrated transposon is still rudimentary, it appears that transposon vectors have the capacity to provide long-term expression of transgenes both in vitro and in vivo.
Considerable effort has been devoted to the development of gene delivery strategies for the treatment of inherited and acquired disorders in humans. A desirable gene therapy approach should 1) achieve delivery of therapeutic genes at high efficiency specifically into the relevant cells, 2) be adaptable to changing needs in terms of vector design, 3) minimize the risk of genotoxicity, and 4) be cost-effective.
Adapting viruses for gene transfer is a popular approach; for example, γ-retroviral and lentiviral vectors are efficient at integrating foreign DNA into the chromosomes of transduced cells and have enormous potential for lifelong gene expression . A major concern of using retroviral vectors is the potential for mutagenic effects at the sites of genomic integration [112–114]. Indeed, insertional mutagenesis has been observed in clinical trials using a retroviral vector for gene therapy of X-linked severe combined immunodeficiency [112, 114, 115]. The clinical use of retroviral vectors can be curtailed because of the limited size of the payload, as multiple or large transgenes compromise the efficiency of viral reverse transcription and packaging. Finally, regulatory issues and the high costs associated with manufacture of clinical-grade retrovirus hamper their widespread translation into clinical practice. An ideal therapeutic vector would combine the favorable attributes of integrating viral vectors (that is, stable chromosomal insertion) while significantly reducing the potential for adverse events. Transposons could potentially offer such an alternative (Figure 2).
The advantage of SB transposon-based gene delivery is that, owing to stable genomic insertion of expression cassettes, it can lead to both long-term and efficient transgene expression in preclinical animal models . Thus, the SB plasmid-based transposon system combines the advantages of viral vectors with those of naked DNA molecules. However, in contrast to viral vectors, transposon vectors can be maintained and propagated as plasmid DNA, which makes them simple and inexpensive to manufacture, an important issue for the implementation of future clinical trials. Further advantages of the SB system include its reduced immunogenicity , no strict limitation of the size of expression cassettes  and improved safety and toxicity profiles [87, 117–119]. Because the transposition mechanism does not involve reverse transcription, DNA-based transposon vectors are not prone to incorporating mutations and can tolerate larger and more complex transgenes, including those containing repeat DNA motifs. Moreover, the use of SB-based gene delivery eliminates the risk of rearrangements of the expression cassette that, as part of a transposing unit of DNA, integrates into chromosomal DNA in an intact form . Compared with retroviral systems, the SB vectors have an inherently low enhancer/promoter activity [117, 118]. Inserting insulator sequences flanking the transcription units of the cargo to prevent accidental trans-activation of promoters of neighboring genes further increased the safety features of the SB system (Figure 3D) . Notably, the transposase can be provided as messenger RNA, thereby reducing the risk of 'rehopping' of the transposon-based vector . Chromosomal integration of SB transposons is precise and random (see above), and no SB-associated adverse effects have been reported [116, 120, 121]. Of note, a precise integration mechanism, random integration pattern and negligible promoter/enhancer activity do not appear to be general features of all recombinase/transposon systems. For example, integration promoted by the bacteriophage-derived PhiC31 system was reported to generate chromosomal rearrangements [122, 123]. The 5' TIR of the piggyBac transposon exhibits significant promoter activity in mammalian cells , and its genomic integration profile resembles that of integrating viral vectors , as described above.
The past few years have seen a steady growth in interest in applying the SB system for the treatment of several conditions including haemophilia A and B [103, 104, 106, 107, 125], junctional epidermolysis bullosa , tyrosinemia I , Huntington disease  sickle cell disease , mucopolysaccharidosis [105, 130], cancer [109, 131] and type 1 diabetes . In addition, important steps have been made towards SB-mediated gene transfer in the lung for potential therapy of α-1-antitrypsin deficiency, cystic fibrosis and a variety of cardiovascular diseases [108, 133]. Thus, the establishment of non-viral, integrating vectors has generated considerable interest in developing efficient and safe vectors for human gene therapy [116, 120, 134–136].
The SB100X hyperactive transposon system yields efficient stable gene transfer after non-viral gene delivery into therapeutically relevant primary cell types, including stem or progenitor cells. For example, the use of the SB100X system yielded robust gene transfer efficiencies into human hematopoietic progenitors [27, 110], mesenchymal stem cells, muscle stem/progenitor cells (myoblasts) and iPSCs . These cells are relevant targets for stem cell biology and for regenerative medicine and gene- and cell-based therapies of complex genetic diseases. Importantly, expression of the SB100X hyperactive transposase did not adversely influence the differentiation or function of these adult stem/progenitor cells, nor was there any evidence of any cytogenetic abnormalities . In the context of iPSC technology, the ability to coax the differentiation of pluripotent stem cells into clinically relevant, transplantable cell types is a key step towards their ultimate use in clinical applications, especially because undifferentiated iPSCs pose an intrinsic tumorigenic risk . It was recently demonstrated that SB transposon-mediated delivery of the myogenic PAX3 transcription factor into iPSCs coaxed their differentiation into MyoD+ myogenic progenitors and multinucleated myofibers , suggesting that PAX3 may serve as a myogenic 'molecular switch' in iPSCs, a finding that has implications for cell therapy of congenital degenerative muscle diseases, including Duchenne muscular dystrophy.
The first clinical application of the SB system is currently ongoing using autologous T cells genetically modified to redirect specificity for B-lineage malignancies . Lymphocytes are a suitable initial platform for testing new gene transfer systems, as there have been hundreds of infusions of clinical-grade T cells genetically modified using viral and non-viral approaches without apparent genotoxicity . The SB transposon tested in the first human application carries a chimeric antigen receptor (CAR) to render the T cells specifically cytotoxic toward CD19-positive lymphoid tumors [141, 142]. The advantage of using the SB system for the genetic modification of T cells includes the reduced cost associated with manufacturing of clinical-grade DNA plasmids compared with recombinant viral vectors. This is particularly important when one considers that trials infusing CAR-positive T cells are only now beginning to demonstrate anti-tumor effects [143, 144]. The higher enzymatic activity of SB100X might enable integration efficiencies comparable with that of retroviral vectors to be achieved for next-generation trials.
Transposon-based technologies have enormous potential to develop powerful genomic tools with the vision of creating a bridge between physiology and genetics and to establish safe and inexpensive protocols for clinical gene transfer. Simple, plasmid-based vectors matched by a corresponding transposase source offer an easy and efficient method for germline transgenesis in laboratory animals and in large animal species for biotechnology. Furthermore, it is now both accessible and practical to generate highly complex libraries of gene knockouts in model species with a view to establishing new models of human disease for the annotation of disease pathways and for therapeutic and pharmaceutical intervention. The recently developed SB100X hyperactive transposon system yields highly efficient stable gene transfer after non-viral gene delivery into therapeutically relevant primary cell types, including stem cells, and thus may facilitate the clinical implementation of ex vivo and in vivo gene therapies. The next phase of preclinical research will focus on further refinement in large animal models to undertake SB-mediated transposition in vivo and to improve the safety profile of SB vectors by target-selected transgene integration into genomic 'safe harbors'. Although it remains to be seen whether the first clinical application of the SB system will result in a therapeutic effect, this trial will help validate the safety of this approach. The ongoing investigations will certainly prompt new ideas and new designs to be developed in this (ever) expanding universe of transposon technologies for genetic and cell engineering.
Work in the authors' laboratories was supported by EU FP6 (INTHER) and EU FP7 (PERSIST and InduStem), grants from the Deutsche Forschungsgemeinschaft SPP1230 'Mechanisms of gene vector entry and persistence', and from the Bundesministerium für Bildung und Forschung (NGFN-2, NGFNplus, iGene, InTherGD and ReGene).
Both authors contributed to drafting, reading and approving the final manuscript.


A minimum 66–94 credits are required for majors in art, depending on your chosen emphasis, including 23–36 upper-division credits.
A minimum 33–36 credits are required for minors in art, depending on your chosen emphasis, including 15–17 upper-division credits.
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This Wireless Hidden Camera for Bathroom is so silent when it works,no doubt it can be a perfect spy camera.The hidden camera is tiny and its position is so secret,you can use it to shoot a lot of exciting videos.It has high definition which is up to 1920X1080, you can get vivid videos without difficulty.Its a mini device that with wifi system which is built inside a unnoticeable shower rack,This Wifi Spy Camera can work anytime and anywhere even when you are at the corner of the world,for example you can watch your baby in USA while you are in England,you just need a WIFI network and iOS/Andriod Smart Cellphone or win10 PC to monitor.This Wifi-Wireless Spy pinhole camera has built-in Wifi base,just download The APP and instruction which is along with the device,quite convenient to use.No wonderful things can avoid this professional spy pinhole camera.
Hidden Spy shower rack Camera with Built in Wifi Antenna System.
This Wireless Hidden Camera for Bathroom operation is fast and convenient, easy to record everything you want.
Item received on time and as described. Its a really useful gadget.
Great product for the price. Video quality adequate to the price. Nice camera to have fun with kite.


The General Block allows you to place and edit text, images and links. It is the most commonly used HotDoodle Block type.
Click the edit doodle icon, relative to the General Block you wish to modify. A simple text editor will open allowing you to insert or edit text. In the text editor window, a pull-down menu will be present with various options to modify font style or size. These menu options should look familiar to you from your word processing software, such as the bold, italic, and underline icons. Other options include adding bulleted lists and modifying text color, text background color, and text alignment. Once text has been inserted or configured, press the Save button.
Note: Choices of fonts and font sizes are based on the cascading style sheet web standard.
To insert a link into text or an image first requires the text or image to be highlighted. Once highlight, click the link icon. A pop-up window will appear where the link destination is to be specified.
If you are linking to content outside your HotDoodle site, then supply the URL in the "Manually enter address" field.
If you are linking to content within your HotDoodle site, then select "link to a page," which will result in a pop-up window displaying a list of pages within your site. Select from a destination within your Site Hierarchy.
If you are wishing to link to a specific location on a page within your HotDoodle site, then select "Link to Content." A pop-up window will appear with blocks marked with a "link to this block" link. Selecting a link will make that particular block the destination on the specified destination page.
Using the General Block, images can be inserted among text. Images can be sourced from an internal link, external link, or your HotDoodle image library.
Lower resolution images are best suited for web pages due to page loading time and monitor capabilities; high resolution images are typically used for print.
In order to be visible on the web, images must be live on a web server. Images can be sourced from your computer or other storage services. In addition, HotDoodle provides the option to stores images on its image manager module.
Images larger than 2 Megabytes (2000 Kilobytes) may fail to upload due to limitations in web browsers and web servers.


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The challenge of managing keys in the education sector
deister UK continues to deliver a reliable key management solution for a diverse and growing city based university.
A combined security and estate management project team at the university identified the challenges of managing keys and user access for this large city campus. The needs and demands of each building and service user were quite different. As part of the education sector, consideration had to be given to staff, students and contractors, alongside location and building purpose. The ability to have the right keys available at the right location, for the right person, was seen to offer cost efficiencies, and would have less demand on staff and their time. It would also lessen the financial and operational risk of either losing or not being able to trace keys when needed. As the on-going support and success of the system was the responsibility of the estates & security team, the factors below were seen as imperative.
• Reliable and low maintenance.
• Professional and prompt manufacturer support.
• Centrally and dynamically managed.
• Ability to adapt, relocate, expand and future proof.
• Managed levels of authorisation.
• Automated reports for departments / buildings.
• Secure and easy override with full disaster recovery.
Modular range of cabinets
"We chose deister electronic as they offered the largest and most modular range of cabinets available. The contactless technology and lifetime guarantee on the keyTag reassured us of the system performance data reliability. deister understood the need to be flexible with such a changing estate, and the built-in disaster recovery system meant that a remote site would always be able to access their keys. As new buildings have come on-line or current buildings repurposed, expansion of the deister system across the University has grown. Other faculties have seen the benefits, and are knowledgeable that it is a trusted system which can be easily supported by the security team. Apart from controlling the primary keys in security, it is helping users and the university to manage a range of buildings for a wide range of departments. The pro-active maintenance and assistance we have received over the last ten years, has helped us to maintain service continuity, and adapt to meet the challenges of an ever evolving University estate."
The reliability of the system continues to deliver cost efficiencies to such an extent, that a second larger phase of cabinet deployment has recently been deployed. The project has been considerate of initial surveys, and the security needs of each department and location. Within the university the deister key management system has become a trusted and effective bolt-on solution, with user friendly software providing flexible and automated management. The modular design of the cabinet has meant older systems haven't gone out of date, and can be easily unified to work with the latest models and operated by the most up to date software. This will assist the security and estates team to continually deliver the challenges of further efficiencies, and the levels.
Project objective
Controlling the primary keys in security, helping users and the university to manage a wide range of buildings.
To manage keys and user access for an estate consisting of several hundred buildings. The ability to have the right keys available at the right location, for the right person.
A user friendly key management system, which saves costs and would have less demand on staff and their time.
The deister key management system has become a trusted and effective bolt-on solution, with user friendly software providing flexible and automated management.


text:
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Currently a blogger and senior editor at the American Conservative, Rod Dreher has served as editor and writer for the New York Post, National Review, The Dallas Morning News, and other publications. His writing focuses on religion in the public square and conservatism. Dreher is the author of several books, including Crunchy Cons (2006), The Little Way of Ruthie Leming (2013), How Dante Can Save Your Life (2015), and The Benedict Option (2017). He lives with his wife Julie and three children in Louisiana.
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Retiring reliably, leaving a legacy or balancing both?
In fall 2010, I posted a blog on the subject of retirement planning: "Is it you … or the kids?" It's been helpful ever since, so I thought I'd revisit that post, and freshen it up with additional ideas.
At the time, I noted how traditional defined benefit (DB) pension plans – and their guaranteed, inflation-adjusted income for life – were becoming increasingly rare, especially in the private sector. The continued demise of DB plans is not all bad news. As nice as they were, they meant that your employer was calling many of your financial planning shots – for better or for worse.
Do you want to leave a substantial legacy? At one end of the spectrum, you can choose to have a greater financial legacy to pass on to your children or charity.
Do you want to live reliably in retirement? At the other end, you may prefer to ensure retirement income sustainability for as long as you live – similar to the kind of stability a traditional DB pension plan provided.
Bottom line, you get to weight the decision one way or the other. As we surmised in our earlier post, this basically means deciding: Is your remaining wealth more for you or the kids?
Legacy Planning – If you want to emphasize leaving a legacy, it stands to reason that your portfolio's average expected return should exceed your withdrawal rate, so inflation doesn't eat away at the balance. This usually means keeping more of your investments working in the markets, while also arranging for a way to take out cash on a regular basis. While this kind of strategy offers you better odds for leaving a bigger legacy, it does not guarantee success. Higher expected returns almost always entail higher risks.
Retirement Sustainability – For emphasizing retirement income sustainability, you'll want to look into a strategy that offers more dependable outcomes … which also translates to lower expected returns. As I described in my earlier post, Dr. Moshe Milevsky and Alexandra MacQueen have co-authored a book entitled Pensionize Your Nest Egg, which describes a strategy for creating your own personal pension-plan-like stream of monthly income. I'm also available for a conversation if I can help you think through your retirement planning.
When should you plan for your "you or your kids" strategy?
There's never a bad time to think about your financial future, but there are a couple of key times for considering what you'd like to achieve in retirement. The most obvious is the "retirement risk zone," 5–7 years in advance of your retirement. It's also worth visiting when you are much younger. If you know in advance that you'd like to build pension-like strategies for retirement, the economics of implementing them in your relative youth can be very attractive.
Again, when shooting for a greater financial legacy or higher income sustainability, remember that there are trade-offs either way. To shoot for greater wealth entails more risk. To keep your money safer implies lower returns. Different factors – such as personal preference, risk tolerance and legacy goals – play a part in which balance makes the most sense for you.
So, is it you or the kids? Ultimately, it's really one or the other.


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MFB sends aid to West Virginia
On Thursday, June 23, in West Virginia, 9 inches of rain fell in 6 to 8 hours, resulting in devastating floods. 26 people were killed, and over 1,200 homes were damaged or destroyed. Midwest Food Bank, a Salvation Army First Responder, was called on to provide aid. 5 semi loads of disaster relief boxes and bottled water have been sent from MFB's Bl...
New division of MFB opening in Phoenix, AZ
Midwest Food Bank is expanding to the southwest with plans to open a food bank in the Phoenix area. A 25,800-square-foot building has been purchased in Gilbert, Ariz., said David Kieser, food bank president and CEO. MFB has considered expansion into Arizona for several years. The Phoenix area location is MFB's eighth site. It should begin providing...
Reactions to fresh produce
It has been six weeks since Midwest Food Bank began getting loads of fresh produce donated by Walmart. We began doing Tuesday and Thursday produce distributions during the weeks we are not doing regular distribution. We'd like to share some of the many positive comments from the agencies we serve: "It was well received. Our families can't always af...
Bloomington Division relocating
Bloomington-based Midwest Food Bank plans to relocate its office and distribution center at 1703 S Veterans Parkway, Bloomington to 2031 Warehouse Drive, Normal. The new location formerly housed the Bloomington-Normal Seating Company. "The last five years has seen an increase of over 130% in the value of food distributed from Midwest Food Ban...
Community steps up
The 7th annual "Give Thanks event is over and done, but the love of the donors and volunteers lingers on in the homes of those who received to food. Nearly 2,300 turkeys and boxes of "fixins" were distributed to families over the course of the two-day event, hosted by Midwest Food Bank and Home Sweet Home Ministries. More than 11,000 individuals we...
State Farm video features MFB volunteer
In an effort to promote volunteerism within their own employees, State Farm created a video focusing on MFB volunteer Jeni Hanson and the work she does here. We are pleased that Jeni and MFB were chosen for this undertaking, and the results are great! To see the amazing video and story with photos, click here.
2016 Volunteers of the Year
Midwest Food Bank Bloomington is happy to announce the recipients of our "2016 Volunteer of the Year Award." The honor goes to Chris Stoffer and Steve Nolan. Volunteers who receive this recognition are selected by MFB management. They meet all of the requirements and responsibilities of being an MFB volunteer and go beyond by seeking other ways to ...
Giving a cup of water
"And whosoever shall give to drink unto one of these little ones a cup of cold water only in the name of a disciple, verily I say unto you, he shall in no wise lose his reward." Matthew 10:42 Midwest Food Bank had an opportunity to give a "cup of cold water" to neighbors in our own community. Late on March 9, a water main broke in Normal, pro...
MFB hires a Chief Financial Officer
Please join us in welcoming Eric Hodel to the Midwest Food Bank team. He has been hired for the new position of Chief Financial Officer. Eric has a BS in Agricultural Engineering from the University of Illinois and has held multiple positions within Caterpillar, most recently as the TTT Product Manager for North America. During Eric's tenure at Cat...
Serving from a new location
Midwest Food Bank is now fully operational at our new location: 2031 Warehouse Road, Normal, IL. Food had gradually been stored here as warehouse space was completed. Offices were moved on Saturday, April 15, and Monday, April 17 marked our first full day here. The Veterans Parkway warehouse seemed huge when we moved in over 11 years ago. Over time...
Golf Outing 2017
Friday, June 16, 2017, was a beautiful day – blue skies and a gentle breeze. The LORD blessed our second annual Golf Outing with excellent weather. Golfers began arriving at 11 am to register. Golfers savored a lunch furnished by Chick-fil-A. After a brief message and a prayer, they were off. The Den at Fox Creek was the perfect course for enjoying...
Food Bank Flier 2017
Midwest Food Bank received a wonderful blessing from Team 224, an offer to host a fundraising bike ride. The first Food Bank Flyer was held on Saturday, June 24, 2017. It was a beautiful summer morning, cool and sunny. Cyclists began arriving at 6:30am to register. The first riders left at 7am. Others registered and left later. There were 20, 40 an...
Disaster Relief for Texas
As a Salvation Army first responder, Midwest Food Bank is answering the call to send disaster relief food boxes to Texas. This request for aid is unique in that it is made for a disaster yet to happen. Harvey is expected to make landfall on the central part of the Gulf coast early Saturday morning as a major hurricane. The storm is expected to stal...
Open House and Ribbon Cutting
On Thursday, September 21, Midwest Food Bank had the blessing of hosting an open house at our new facility. Guests followed a map for a self-guided tour. At the end of the tour, delicious hors d'oeurvres were ready to enjoy. At 4pm, everyone gathered for a McLean County Chamber of Commerce ribbon cutting. As with every MFB endeavor, volunteers play...
Caring Community Gives Thanks
Midwest Food Bank and Home Sweet Home Ministries completed another successful year of Give Thanks. With the generous support of the community, we collected enough turkeys and meal items to impact nearly 10,000 individuals. Over 200 volunteers distributed the turkeys and meal boxes on November 18th and 20th at Midwest Food Bank. With the combined di...
Partnership with Jackie Joyner-Kersee
Midwest Food Bank had a very special visitor today. Jackie Joyner-Kersee, one of the most decorated female athletes in Olympic track and field history, was here! As the founder of the Jackie Joyner-Kersee Foundation in her hometown of East St. Louis, Jackie and MFB founder, David Kieser, are working out the details of a partnership where MFB can su...
A little spark
An old proverb says a little spark can kindle a great fire. A classroom of of first graders at Sugar Creek Elementary provided that little spark. The fire grew and warmed many people. Miss Lyons first grade class watched a video of the devastation Hurricane Harvey caused in Texas. The classs began brainstorming ways they could offer help to the peo...
Midwest Food Bank Bloomington-Normal is pleased to announce our 2017 Volunteers of the Year. Out of the thousands of volunteers who gave of their time and talent last year, these three are being recognized for going above and beyond the call of duty. They are Gretchen Dean, Charles Sang and Jeff Snyder. Get to know them better: Gretchen, already vo...
From volunteer to employee
Midwest Food Bank Bloomington-Normal is happy to announce the hiring of Jeni Hanson as Warehouse Manager. Jeni's first day in her new position was January 23. But it certainly wasn't her first day at MFB. Already a valued volunteer, Jeni is well-known to the regulars here. She was very sensitive to the mission of MFB from the start. She soon became...
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We arrived in Pigeon Forge on a Monday and stayed through Thursday. Our accommodations were at a place called Riverstone Resort and Spa right off of Dollywood Lane. It was a spectacular place to stay. It is not a hotel/motel type place but is made up of a series of buildings with 2 -4 room condos. They either overlook a river or a golf course. Our condo was a two bedroom that overlooked the golf course from our back patio. Both bedrooms had king beds with the smaller room sharing a bathroom with the other areas but the main room had a huge walk-in closet with a bathroom of its own. The back patio/balcony was entered either from the main bedroom or the living room. The living room had a fireplace, huge tv, two couches with one being a queen pull-out bed. There was full size table that sat 6 people and a full size kitchen complete with dishwasher. The best thing I thought was the screened in porch area. It faced the front of the resort. Normally you get off the elevator and the doors would be right up front on the walkway. Not this place. Our condo had a screened in porch that you would enter before getting to the door of the condo. Plus the kitchen had a huge sliding door right into the porch area. I loved all three nights we stayed there. It was quiet even though it was almost full of people staying there. It also had above ground parking or you could use the parking garage under each building. There was an indoor heated pool, spa treatments available, a large playground for the kids, and a lazy river but it was closed for the season still. I would love to stay there again if we were to go for more than two days on another visit. I don't have any of my own pictures but will post a few off the website along with the address for it.
We also bought our Dollywood tickets from here when we reserved and paid for the room online. Very easy and quick to do.
We didn't get to do too much while there. We spent Tuesday driving to Cades Cove in the national park then returned to Pigeon Forge. After that we spent almost six hours, yes I said six hours, at one of the go-cart places. We were tired to the max after returning to the condo sometime after 10 o'clock.
Our next day was spent all day at Dollywood. Make sure you check out their website before you go. It will tell all the important things like parking fees, opening times, and will help you plan the things you want to do. We were lucky the day we went in that it was only the second day open for the season so there was no parking fees. Yay! I wouldn't worry about how far out you have to park because they run plenty of shuttles from all areas. There might be a slight wait for one but it is worth not having to walk the long distance from your vehicle to the entrance. Plus you get a nice breeze going during the ride. Be prepared for lots of walking and I do mean lots of walking.
I don't know if I would want to do it again but it was fun. I would recommend going before it got too hot but just be ready for some cold water if you do the water rides.
Since we didn't get to do any of the other wonderful things in Pigeon Forge, the kids and I set out when it was dark and took pictures when things were all lit up.
Make sure you get online to the Pigeon Forge tourism website and order a brochure/book from them. We ordered one from Pigeon Forge and Gatlinburg.