Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

Rare germline variants in PALB2 and BRCA2 in familial and sporadic chordoma.

Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

Functional evaluation of five BRCA2 unclassified variants identified in a Sri Lankan cohort with inherited cancer syndromes using a mouse embryonic stem cell-based assay.

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.

Interaction with PALB2 Is Essential for Maintenance of Genomic Integrity by BRCA2.

Human breast cancer susceptibility gene, BRCA2, encodes a 3418-amino acid protein that is essential for maintaining genomic integrity. Among the proteins that physically interact with BRCA2, Partner and Localizer of BRCA2 (PALB2), which binds to the N-terminal region of BRCA2, is vital for its function by facilitating its subnuclear localization. A functional redundancy has been reported between this N-terminal PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2, which undermines the relevance of the interaction between these two proteins. Here, we describe a genetic approach to examine the functional significance of the interaction between BRCA2 and PALB2 by generating a knock-in mouse model of Brca2 carrying a single amino acid change (Gly25Arg, Brca2G25R) that disrupts this interaction. In addition, we have combined Brca2G25R homozygosity as well as hemizygosity with Palb2 and Trp53 heterozygosity to generate an array of genotypically and phenotypically distinct mouse models. Our findings reveal defects in body size, fertility, meiotic progression, and genome stability, as well as increased tumor susceptibility in these mice. The severity of the phenotype increased with a decrease in the interaction between BRCA2 and PALB2, highlighting the significance of this interaction. In addition, our findings also demonstrate that hypomorphic mutations such as Brca2G25R have the potential to be more detrimental than the functionally null alleles by increasing genomic instability to a level that induces tumorigenesis, rather than apoptosis.

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn.

Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt-Hogg-Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.

Selenophosphate synthetase 1 is an essential protein with roles in regulation of redox homoeostasis in mammals.

Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogues, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyses the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1-knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by embryonic day (E)8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione transferase Omega 1 (GSTO1). To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma (EC) cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Furthermore, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homoeostasis and controlling cell growth.

Fgf3-Fgf4-cis: A new mouse line for studying Fgf functions during mouse development.

The fibroblast growth factor (FGF) family consists of 22 ligands in mice and humans. FGF signaling is vital for embryogenesis and, when dysregulated, can cause disease. Loss-of-function genetic analysis in the mouse has been crucial for understanding FGF function. Such analysis has revealed that multiple Fgfs sometimes function redundantly. Exploring such redundancy between Fgf3 and Fgf4 is currently impossible because both genes are located on chromosome 7, about 18.5 kb apart, making the frequency of interallelic cross-over between existing mutant alleles too infrequent to be practicable. Therefore, we retargeted Fgf3 and Fgf4 in cis, generating an Fgf3 null allele and a conditional Fgf4 allele, subject to Cre inactivation. To increase the frequency of cis targeting, we used an F1 embryonic stem cell line that contained 129/SvJae (129) and C57BL/6J (B6) chromosomes and targeting constructs isogenic to the 129 chromosome. We confirmed cis targeting by assaying for B6/129 allele-specific single-nucleotide polymorphisms. We demonstrated the utility of the Fgf3(Δ)-Fgf4(flox)-cis mouse line by showing that the caudal axis extension defects found in the Fgf3 mutants worsen when Fgf4 is also inactivated. This Fgf3(Δ)-Fgf4(flox)-cis line will be useful to study redundancy of these genes in a variety of tissues and stages in development.

Essential role of the zinc finger transcription factor Casz1 for mammalian cardiac morphogenesis and development.

Chromosome 1p36 deletion syndrome is one of the most common terminal deletions observed in humans and is related to congenital heart disease (CHD). However, the 1p36 genes that contribute to heart disease have not been clearly delineated. Human CASZ1 gene localizes to 1p36 and encodes a zinc finger transcription factor. Casz1 is required for Xenopus heart ventral midline progenitor cell differentiation. Whether Casz1 plays a role during mammalian heart development is unknown. Our aim is to determine 1p36 gene CASZ1 function at regulating heart development in mammals. We generated a Casz1 knock-out mouse using Casz1-trapped embryonic stem cells. Casz1 deletion in mice resulted in abnormal heart development including hypoplasia of myocardium, ventricular septal defect, and disorganized morphology. Hypoplasia of myocardium was caused by decreased cardiomyocyte proliferation. Comparative genome-wide RNA transcriptome analysis of Casz1 depleted embryonic hearts identifies abnormal expression of genes that are critical for muscular system development and function, such as muscle contraction genes TNNI2, TNNT1, and CKM; contractile fiber gene ACTA1; and cardiac arrhythmia associated ion channel coding genes ABCC9 and CACNA1D. The transcriptional regulation of some of these genes by Casz1 was also found in cellular models. Our results showed that loss of Casz1 during mouse development led to heart defect including cardiac noncompaction and ventricular septal defect, which phenocopies 1p36 deletion syndrome related CHD. This suggests that CASZ1 is a novel 1p36 CHD gene and that the abnormal expression of cardiac morphogenesis and contraction genes induced by loss of Casz1 contributes to the heart defect.

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

The folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dubé syndrome is required for murine B-cell development.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by cutaneous fibrofolliculomas, pulmonary cysts, and kidney malignancies. Affected individuals carry germ line mutations in folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney cancer, FLCN has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germ line deletion of Flcn results in early embryonic lethality in animal models. Here, we describe mice deficient in the newly characterized folliculin-interacting protein 1 (Fnip1). In contrast to Flcn, Fnip1(-/-) mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is entirely independent of mTOR activity. We show that this developmental arrest results from rapid caspase-induced pre-B cell death, and that a Bcl2 transgene reconstitutes mature B-cell populations, respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1(-/-) mice. Our studies thus demonstrate that the FLCN-FNIP complex deregulated in BHD syndrome is absolutely required for B-cell differentiation, and that it functions through both mTOR-dependent and independent pathways.

Regulation of trafficking of activated TrkA is critical for NGF-mediated functions.

Upon activation by nerve growth factor (NGF), TrkA is internalized, trafficked and sorted through different endosomal compartments. Proper TrkA trafficking and sorting are crucial events as alteration of these processes hinders NGF-mediated functions. However, it is not fully known which proteins are involved in the trafficking and sorting of TrkA. Here we report that Nedd4-2 regulates the trafficking of TrkA and NGF functions in sensory neurons. Depletion of Nedd4-2 disrupts the correct sorting of activated TrkA at the early and late endosome stages, resulting in an accumulation of TrkA in these compartments and, as a result of the reduced trafficking to the degradative pathway, TrkA is either reverted to the cell surface through the recycling pathway or retrogradely transported to the cell body. In addition, Nedd4-2 depletion enhances TrkA signaling and the survival of NGF-dependent dorsal root ganglion neurons, but not those of brain-derived neurotrophic factor-dependent neurons. Furthermore, neurons from a knock-in mouse expressing a TrkA mutant that does not bind Nedd4-2 protein exhibit increased NGF-mediated signaling and cell survival. Our data indicate that TrkA trafficking and sorting are regulated by Nedd4-2 protein.

Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9.

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.1.

Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant.

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females.

RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution.

BRCA2-DSS1 interaction is dispensable for RAD51 recruitment at replication-induced and meiotic DNA double strand breaks.

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

An inducible and reversible mouse genetic rescue system.

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.