You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: An Inducible and Reversible Mouse Genetic Rescue System}
Article:
Abstract:
Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated( TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem( ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E( ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.
Introduction:
In the post-genome era, a major challenge is deciphering the function of thousands of newly identified genes. One of the main approaches for studying gene function involves inactivation of genes in cells or animals using random( chemical or insertional) mutagenesis or gene targeting. A common problem with these methods stems from the fact that the gene of interest is usually mutated throughout the animal's life. As a result, 1) in many cases the mutation leads to embryonic or neonatal lethality, precluding the assessment of the gene's function in later life; 2) in viable mutants interpretation of observed phenotypes is often complicated by the inability to distinguish the direct effects of the gene loss at the time of observation from the results of developmental abnormalities caused by the gene loss earlier in life; 3) in still other cases, life-long absence of a gene product causes compensatory adjustments of activities of other genes precluding the elucidation of the function of the gene of interest. Conditional knockout and gene expression technologies, such as the Cre/lox-mediated tissue-specific knockout[1] and the tetracycline( Tet) regulated transcriptional activation system[2], can regulate gene expression in a more spatially and temporally controlled fashion. However, these technologies are often laborious to establish and the results are frequently variable. Here we report the development of a system that provides for the inducible and reversible gene inactivation in the mouse and can also be readily scaled up for high-throughput applications. The iKO system is a binary approach based on the Tet-dependent regulatory technology. It involves the combination of two mouse lines – a KO line that expresses the Tet-transactivator( tTA or rtTA) in place of the gene of interest, and a TIGRE( for tightly regulated) line that contains the gene of interest under the control of the Tet-responsive element( TRE) at a predetermined genomic locus. It has the advantage of, 1) ability to turn genes on or off at will by adding or removing doxycycline( Dox) at any time during the animal's life, thus minimizing embryonic lethality, developmental effects, and compensatory effects; 2) high degree of regulation to any gene inserted at the TIGRE locus, which has been selected to confer minimal basal expression and high inducibility, and to insert any gene of interest in a single step by Cre/loxP recombination; 3) efficiency; the design allowing streamlined production of both KO and TIGRE mice makes it possible to generate iKOs for a large number of genes in a cost-effective manner; 4) flexibility; KO and TIGRE lines can be engineered independently and combined in numerous ways, making a wide range of applications possible. As a proof of concept, we report the characterization of an iKO of the apolipoprotein E gene( ApoE iKO). ApoE plays a key role in regulating cholesterol metabolism and atherosclerosis progression. ApoE KO mice develop hypercholesterolemia and atherosclerosis that closely resemble the human conditions and are rapidly reversed when APOE protein is supplied[3],[4],[5],[6],[7],[8]. Thus, inducible and reversible regulation of ApoE expression could result in rapid physiological changes, which in turn can help assess the iKO technology. Furthermore, the phenotype of ApoE deficiency is quantifiable and very sensitive to leaky expression, allowing for the evaluation of the stringency of gene regulation by iKO technology[9]. Here we demonstrate that in the ApoE iKO mice, ApoE gene expression, as well as blood cholesterol levels, is tightly controlled by Dox. In the presence of Dox, ApoE is expressed and the cholesterol levels are low; in its absence, the reverse is observed. Furthermore, on examination of aortic atherosclerosis in the ApoE iKO mice we found that Dox treatment before the onset of atherosclerotic lesions completely prevented lesion formation and Dox treatment after extensive lesions had already formed resulted in regression of the lesions. These results demonstrate the reversibility of the iKO, leading to phenotype switching within the same animal. ApoE iKO is also useful in its own right as a novel model system for the study of molecular mechanisms underlying atherosclerosis progression and regression.
Discussion:
We have developed a reversible and inducible rescue system for gene KO in mice and have applied this method into the ApoE gene. Our system complements existing inducible gene expression approaches and provides certain advantages. The tamoxifen-dependent Cre-ERT2[19],[20] recombination can drive inducible knockout of the endogenous gene, however, it is irreversible and the efficiency of tamoxifen inducibility throughout the body is yet to be demonstrated. The usefulness of the Tet-inducible system[21],[22] is critically dependent on the tightness of transcription induction and suppression. When either the Tet-transactivator or the target gene is randomly integrated into the genome, they are subjects to positional effects[23]. It is often necessary to screen through multiple transgenic lines every time a new transgene is introduced. Attempts to target both tTA and TRE together into an endogenous gene locus to achieve inducible activation and inactivation were successful in a few cases[24],[25],[26], but this method is not applicable to most other genes as the TRE is easily subject to activation from nearby enhancers independent from tTA. Our iKO technique utilizes each gene's own promoter to direct the expression of transcriptional activators, e. g. rtTA and tTA. Therefore it is not limited to certain tissues and available tissue-specific promoter driven transgenic lines. It could be applicable to any gene in any tissue. Dox-regulated expression can be turned on and off rapidly, i. e. within a few days, and at any time in development or adult. It also allows analysis of the effects of gene inactivation in the same animal. The TIGRE locus has been selected to confer little or no basal transgene expression throughout the body while maintaining high inducibility, enabling stringent control of the on/off switching of the target gene. Our system also has the flexibility to allow for further improvements. For example, in some cases it may be more desirable to put the genomic copy of the gene under TRE promoter into the TIGRE locus instead of the cDNA to more precisely mimic the expression of the endogenous gene. Also, the IRES we have used to drive rtTA translation may not work uniformly well in all tissues, and can be replaced by other approaches such as using the viral 2A-like sequences for bicistronic translation[27] or direct targeting of tTA/rtTA into the ATG start codon of the endogenous gene. Unique chromosomal loci for predicted gene expression provide fundamental tools for genetic studies. The most widely used is the ROSA26 locus[28] in which ubiquitous gene expression is achieved by endogenous promoter activity of this locus. The TIGRE loci identified in this study allow a different mode of gene regulation – they were selected from hundreds of insertion sites for tight gene regulation by an exogenous promoter. Therefore, the TIGRE loci offer a platform for easy insertion of any gene in a tightly regulated locus, applicable to not only the tetracycline system but also other gene expression systems utilizing exogenous promoters such as constitutively active promoters, tissue specific promoters, or other inducible promoters regulated by reagents such as ecdysone[29], mifepristone[30] and streptogramin[31]. Recently a similar approach was also applied to a human fibrosarcoma cell line to pre-screen optimal integration sites for transgenes[32]. In addition, here we demonstrated that the stringency of the regulation at TIGRE loci is further enhanced by the incorporation of insulators, i. e. basal expression level was reduced to less than one luciferase molecule per cell without impairing inducibility( Figure 4C, D). Insulators were also shown previously to improve inducibility of randomly integrated TRE-reporters[33]. It should be noted that in our study the insulator effect was limited in many other loci( Figure 4A versus 4B), demonstrating the uniqueness of the TIGRE loci. The genomic location of the T1 TIGRE locus, which was most extensively used in our study including the ApoE iKO mice, was determined( Figure 5). Further manipulation of this locus would be possible to expand its application. The stringent gene regulation is demonstrated in the ApoE iKO mice. It is known that regulation of blood cholesterol levels is very sensitive to the plasma ApoE protein levels. Even with the production of 3% of wild-type level of ApoE protein, the hypercholesterolemia and atherosclerosis phenotypes of the ApoE KO mice can be reversed[9]. Given this high sensitivity, the fact that our ApoE iKO mice in the uninduced state( i. e. in the absence of Dox) exhibit similarly high levels of cholesterol compared to ApoE KO mice indicates that there is hardly any expression of functional APOE. Reversing the KO at will is a particularly powerful approach in atherosclerotic regression studies. Dox-treatment of ApoE iKO mice results in expression of ApoE, marked reduction of plasma cholesterol levels, and regression of aortic atherosclerotic lesions. These findings are consistent with previous studies showing that aggressive lipid lowering or expression of ApoE can induce regression of pre-existing atherosclerotic lesion[7],[8],[34],[35]. It is becoming increasingly clear that lesion regression is regulated by a complex interplay between lipids, inflammation and the immune system[35]. The ApoE iKO mice will allow detailed studies on the roles of specific genes in these complex interactions. The binary nature of the iKO system is inherently simple, with the KO line serving dual roles: it could be used as a constitutive KO or combined with the TIGRE line to produce an inducible and reversible iKO. The 10-million clone ES cell library we utilized[18] has been estimated to contain insertional mutations for >90% of genes, and individual ES clones with retroviral insertions in a specific target gene can be rapidly identified through a PCR pooling strategy and subsequently isolated in a streamlined process. The identification and modification of the TIGRE locus allows rapid insertion of any gene of interest via co-transfection with Cre. Therefore, each component of the binary system, the KO or the TIGRE line, is amenable for high-throughput production to generate inducible and reversible KOs for a large number of genes. It should be noted that the iKO system may not be applicable in certain situations where highly stringent gene regulation is required. For example, even though the system had enough stringency in low basal activity and high induction, the induced gene expression level is usually not exactly the same as the endogenous gene's level and this could be a problem for genes that are highly sensitive to gene dosage effect or show haploid deficiency. The kinetics of the system( days) may be also too slow for some developmental problems where transient expression of developmental genes are critical, although it should be noted that it could still work well in carefully thought-out developmental studies( as in[24]). In addition, it has been reported that rtTA often can not induce sufficient gene expression in the brain, at least partially due to developmental inactivation of the TRE promoter in neurons[36]. It appears that the tTA( Tet-off) system is better suited for the use in brain[14], as substantial β-gal induction was observed in brain with tTA( Figure 4F). Our results of the ApoE iKO mice and the quantitative data using lacZ and luciferase reporters suggest that the iKO system could be a useful tool in addressing a variety of biological questions. The two components of the iKO system can be independently modified, and pairing of their different forms can generate numerous combinations. In KO lines, genes with unique expression patterns are tagged with a transcription transactivator, which can control, in an inducible fashion, the expression of a variety of genes derived from TIGRE lines, enabling a number of additional applications in specific types of tissues or cells. Those include: 1) introducing mutant forms of the target gene into the TIGRE locus for better functional probing of different domains, splice variants, post-transcriptional modifications( e. g. phosphorylation), or modeling human diseases; 2) humanizing target genes by placing a human ortholog of the mouse gene under TRE control, which can facilitate drug efficacy studies; 3) placing a cytotoxic gene under TRE control to allow inducible cell-type specific ablation; 4) introducing a marker gene such as GFP, protein interacting probe, or transneuronal tracer, into the TIGRE locus for cell-type specific tagging, functional analysis, isolation of specific cell population, or mapping neuronal networks; 5) combining with recently developed RNAi techniques[37],[38],[39] to down-regulate any genes of interest in a tissue-specific and inducible manner; 6) re-engineering the TIGRE locus to place a TRE-driven Cre, and combining it with a tissue-specific rtTA or tTA line and floxed target genes to achieve inducible region-specific gene knockout[40].
We describe a technology for the creation of inducible and reversible gene inactivation in mice . It combines two genetically modified mouse lines: a knock-out line with a tetracycline transactivator replacing the endogenous target gene , and a line in which a tetracycline-inducible cDNA of the target gene has been inserted into a specific genomic locus . A critical component of this system is the unique chromosomal loci we have identified and engineered that offer a platform for easy insertion of any gene of interest for tightly controlled expression . Because of its simple binary nature , allowing independent modification of each of the two components and possibility of use in a high-throughput mode , we believe that our system will be useful for multiple applications , such as introducing mutant or humanized form of the target gene as well as functional manipulating tools . We have applied this technology to the Apolipoprotein E ( ApoE ) gene and have demonstrated that: a ) the expression of ApoE is strictly dependent on the presence of doxycycline , a tetracycline group antibiotic , in the mouse diet , b ) in the absence of doxycycline ( ApoE repressed ) atherosclerotic plaques are formed , confirming the importance of ApoE in the process , and c ) upon re-induction of ApoE in the animals with doxicyclin , atherosclerosis regressed .