The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

A Mayer

Adam J Bogdanove

Albert W Cheng

Chikdu S Shivalila

D Pirottin

Daniel B Dadon

Daniel F Voytas

David C Page

DY Guschin

G Grant Welstead

GG Welstead

H Skaletsky

Haoyi Wang

J Rohozinski

JC Miller

JF Hughes

KB Shpargel

LL Washburn

MJ Moscou

P Koopman

R Lovell-Badge

Rudolf Jaenisch

S Mazeyrat

Styliani Markoulaki

T Cermak

Tatyana Pyntikova

Yueh-Chiang Hu

Nature Biotechnology

PubMed

Crossref

TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes – Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome. The Y chromosome has unique structure and gene content and has evolved into a chromosome that is highly specialized for male sex differentiation and fertility1, 2. A comprehensive approach to study the function of Y-linked genes in mice is needed to unravel the biology of the Y chromosome. Perhaps due to the unique structural features of the Y chromosome, conventional gene targeting strategies in mESCs to generate mutations in Y-linked genes have been unsuccessful. Therefore, our understanding of the functions of murine Y-linked genes is limited to insights gained from studies of mice that carry spontaneous deletions, random gene trap insertions or autosomal transgenes3-5. Although two Y-linked knockout mESC clones and one transgenic mouse line have been generate

Yueh-chiang Hu

G. Grant Welstead

Albert W

Chikdu S. Shivalila

Daniel B. Dadon

Daniel F

Adam J. Bogdanove

CiteSeerX

Wang, Haoyi

Hu, Yueh-Chiang

Markoulaki, Styliani

Welstead, G Grant

Shivalila, Chikdu Shakti

Pyntikova, Tatyana

Dadon, Daniel Benjamin

Voytas, Daniel F.

Bogdanove, Adam J.

Jaenisch, Rudolf

Welstead, G. Grant

Cheng, Albert Wu

Page, David C

DSpace@MIT

TALEN-mediated editing of the Mouse Y ChromosomeHaoyi Wang1,7, Yueh-Chiang Hu1,2,7, Styliani Markoulaki1, G. Grant Welstead1, Albert W.Cheng1,4, Chikdu S. Shivalila1,3, Tatyana Pyntikova1,2, Daniel B. Dadon1,3, Daniel F.Voytas5, Adam J. Bogdanove6, David C. Page1,2,3,8, and Rudolf Jaenisch1,3,81 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA2 Howard Hughes Medical Institute3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts,USA4 Computational and Systems Biology Program, Massachusetts Institute of Technology,Cambridge, Massachusetts, USA5 Department of Genetics, Cell Biology and Development, University of Minnesota, Saint Paul,Minnesota, USA6 Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York,USAAbstractThe functional study of Y chromosome genes has been hindered by a lack of mouse models withspecific Y chromosome mutations. We used transcription activator-like effector nuclease(TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice withtargeted gene disruptions and insertions in two Y-linked genes – Sry and Uty. TALEN-mediatedgene editing is a useful tool for dissecting the biology of the Y chromosome.The Y chromosome has unique structure and gene content and has evolved into achromosome that is highly specialized for male sex differentiation and fertility1, 2. Acomprehensive approach to study the function of Y-linked genes in mice is needed tounravel the biology of the Y chromosome. Perhaps due to the unique structural features ofthe Y chromosome, conventional gene targeting strategies in mESCs to generate mutationsin Y-linked genes have been unsuccessful. Therefore, our understanding of the functions ofmurine Y-linked genes is limited to insights gained from studies of mice that carryspontaneous deletions, random gene trap insertions or autosomal transgenes3-5. Althoughtwo Y-linked knockout mESC clones and one transgenic mouse line have been generatedusing an insertional targeting strategy (a method causing DNA duplications around thetargeting site)6, 7, no mouse with targeted gene knockout and knock-in on the Ychromosome has been reported. Here we report using TALEN-mediated gene editing8, 9 to8 Correspondence should be addressed to David C. Page [dcpage@wi.mit.edu] or Rudolf Jaenisch [jaenisch@wi.mit.edu].7These authors contributed equally to this work.Author contributionsHW, YCH, DCP, and RJ designed the experiments and wrote the manuscript. HW, DFV, and AJB designed TALENs. HW generatedand tested TALENs. HW preformed targeting experiments. CSS and DBD assisted with Southern blot analysis. SM performedtetraploid complementation experiments. YCH and GGW maintained mutant mouse colonies and performed animal-relatedexperiments. TP performed FISH analysis. AWC performed the off-target analysis. HW, YCH, and GGW analyzed the data.Competing financial interestsThe authors declare no competing financial interests.Published as: Nat Biotechnol. 2013 June ; 31(6): 530–532.HHMI Author ManuscriptHHMI Author ManuscriptHHMI Author Manuscriptefficiently manipulate genes on the mouse Y chromosome and produce mice with mutationsin two different Y-linked genes.We first engineered TALENs (Fig. 1A, Supplementary Table 1) directed against the mouseSry gene. Sry has a well-defined function in testis determination and is therefore anappropriate target for proof-of-principle experiments. Two pairs of TALENs were generatedto target the high mobility group (HMG) DNA binding domain of Sry. We used theSurveyor assay10 to test the nuclease activity of these TALENs on the Sry gene in V6.5 XYmESCs. TALEN pairs 1 and 2 showed gene modification efficiencies of 15% and 20%,respectively (Supplementary Fig. 1). Following TALEN transfection, mESCs were plated atlow density without selection, and clones were picked and genotyped by Southern blotanalysis. Because Sry TALEN pair 2 cleaves a region that includes a BsaJI site, successfullytargeted clones could be identified by a loss of the BsaJI site, using Southern blot analysis(Fig. 1A and B). In three independent targeting experiments we screened 200 mESC clonesand obtained five targeted clones. Each clone harbored a single Sry gene deletion rangingfrom 11 to 540 bp (Fig. 1A). Four of the five targeted mESC clones retained a normalchromosome configuration (Supplementary Fig. 2).To generate mice carrying a targeted mutation (tm) of the Sry gene, Srytm1 (41-bp deletion),Srytm2 (11-bp deletion), and Srytm4 (540-bp deletion) mESCs were injected into tetraploidblastocysts, which were transferred to pseudopregnant females. Embryos were examined atE14.5 (12 days after transfer) and pups were delivered by Caesarian section (17 days aftertransfer). As expected, all E14.5 embryos (n=7) and full-term pups (n=17) that we derivedfrom these three mESC clones displayed sex-reversal such that chromosomal (XY) maleswere anatomically female (Fig. 1C and D). We did not observe any evidence of testiculardifferentiation in either embryos or pups. Adult Sry-targeted mice (anatomic females)showed reduced fertility, but transmitted the Sry-mutated Y chromosome to offspring (Fig.1D and Supplementary Table 2). These data confirm a previous report of anatomic sexreversal in Srydl1Rlb mice carrying a spontaneous 11-kb deletion encompassing the Srylocus3. To confirm that sex reversal was caused by the Sry mutation and not other geneticchanges, we crossed Srytm1 females with Srydl1Rlb;Tg(Sry)2Ei males, a previously describedSry transgenic line11, to produce Srytm1;Tg(Sry)2Ei progeny. Consistent with the previousreport12, pups with the Sry mutation that also carried the Sry transgene (Srytm1;Tg(Sry)2Ei)developed as anatomic males (Fig. 1D and Supplementary Table 2), supporting theconclusion that the anatomic sex reversal observed in Srytm1 mice was due to the absence ofSry.To test for potential non-specific mutations induced by introduction of the TALENs, wepredicted the top 15 potential off-targets of TALEN pair 2 (Supplementary Table 3) usingthe position weight matrix (PWM) of natural TAL effectors13 (Materials and Methods). WePCR-amplified and sequenced these 15 loci in Srytm1, Srytm2, and Srytm4 mESC clones andfound no mutations.To further establish the utility of TALEN-mediated targeting for the Y chromosome weproduced a GFP knock-in allele at the Sry locus. We introduced TALEN pair 2, along with adonor construct that contained a promoterless GFP and a puromycin selection markerflanked by short homologous arms (700 and 385 bp for 5’ and 3’ arms, respectively), intoV6.5 mESCs (Fig. 1E). After transfection, mESCs were selected with puromycin andgancyclovir. In three independent experiments we screened 300 mESC clones and obtainedthree correctly targeted clones, all of which were confirmed by Southern blotting, PCRgenotyping (Fig. 1F and Supplementary Fig. 3) and sequencing (data not shown). Since theSry promoter drives GFP, we tested whether its expression recapitulates endogenous Sryexpression. We performed semi-quantitative RT-PCR to detect GFP transcript levels inWang et al. Page 2Nat Biotechnol. Author manuscript; available in PMC 2013 December 01.HHMI Author ManuscriptHHMI Author ManuscriptHHMI Author Manuscriptvarious embryonic tissues. We dissected E12.0 embryos generated by tetraploidcomplementation and found that they were all anatomically female, indicating Sry geneinactivation. We then confirmed that GFP was expressed in the gonads and brain, but not inother tissues (Fig 1G), consistent with previous reports12, 14. The generation of Sry-GFPknock-in mice demonstrates the feasibility of insertion of a transgene into a specific locus onthe Y chromosome.Successful manipulation of the Sry gene prompted us to test whether the same targetingstrategies could be applied to other Y-linked genes with similar efficiency. We chose Uty asthe second target, because unlike Sry, Uty is expressed in mESCs5, allowing to assay theeffect of targeting. We designed two pairs of TALENs that target the first exon of Uty (Fig.2A and Supplementary Table 1). Although the TALEN pair used to target Uty had a lowefficiency of cutting when tested in a Surveyer assay, we were able to isolate one correctlytargeted clone (Utytm1 that contains a 167-bp insertion derived from the TALEN expressionplasmid) out of 192 mESC clones picked. To improve the likelihood that the tested mESCclones had received the TALEN pair, we co-transfected Uty TALEN pair 2 along with apuroR-containing plasmid into mESCs, and selected for cells with transient puromycinresistance for two days. Drug selection dramatically increased the targeting efficiency from~0.5% to ~9% (five of 56 clones were targeted), as confirmed by Southern blot analysis(Fig. 2B). We then derived mice from Utytm1 mESCs through tetraploid complementation.The Utytm1 mice were viable and fertile. When mated with Utx heterozygous mutantfemales15, no Utxtm/Utytm1 double mutant offspring were obtained, while Utx and Utysingle hemizygous-mutant males were born (Supplementary Table 4). These results suggestthat Utx and Uty function redundantly in embryonic development, which is consistent withthe previously reported phenotypes of gene trap-derived Utx and Uty mutant animals5.Using a similar strategy we generated an Uty-GFP knock-in allele by introducing UtyTALEN pair 2 and a donor construct containing a promoterless GFP and a puromycinselection marker flanked by short homologous arms (832 and 889 bp for 5’ and 3’ arms,respectively) into V6.5 mESCs (Fig. 2C). From three independent experiments, we screened300 mESC clones and obtained six correctly targeted clones in total (~2% targetingefficiency), all confirmed by Southern blotting, PCR genotyping (Fig. 2D andSupplementary Figs. 4 and 5), and sequencing (data not shown). Because GFP is fused in-frame with the first 13 codons of Uty, all correctly targeted mESC clones were GFP positive(Fig 2E and F) as expected. We then generated animals from these Uty-GFP mESCs throughtetraploid complementation. We found that GFP was undetectable by eye in embryos andnewborn pups, which is consistent with a previous report that Uty is expressed at a low levelin mouse embryos5. However, we were able to detect low-level GFP expression in mouseembryonic fibroblasts (MEFs) derived from the E13.5 Uty-GFP embryos using flowcytometry (Fig. 2F). This was confirmed by quantitative RT-PCR analysis, which detected amuch lower expression level of the Uty-GFP transcript in MEFs compared to mESCs (Fig.2G). To determine whether this differential expression is characteristic of the wild-type Utylocus, we compared Uty transcript levels in wild-type MEFs and mESCs and found a similarexpression pattern (Fig. 2G).We demonstrate that both Sry and Uty can be efficiently targeted by TALEN-mediated geneediting strategies, enabling the generation of mice carrying Y-linked gene mutations. Thesystem described here provides a novel and general approach for genetic manipulation of theY chromosome, which has not been possible with conventional gene targeting approaches.Thus, TALEN-mediated gene editing will allow the study of Y-chromosome biology bygenetic manipulation in mice and other species.Wang et al. Page 3Nat Biotechnol. Author manuscript; available in PMC 2013 December 01.HHMI Author ManuscriptHHMI Author ManuscriptHHMI Author ManuscriptSupplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe thank Kibibi Ganz and Ruth Flannery for support with animal care and experiments. We thank Jaenisch andPage lab members for helpful discussions on the manuscript. AWC is supported by a Croucher scholarship. DCP,YCH, and TP were supported by Howard Hughes Medical Institute. RJ, HW, SM, GGW, GSS, and AWC weresupported by National Institutes of Health Grants R37-HD045022 and R01-CA084198.References1. Skaletsky H, et al. Nature. 2003; 423:825–837. [PubMed: 12815422]2. Hughes JF, et al. Nature. 2012; 483:82–86. [PubMed: 22367542]3. Lovell-Badge R, Robertson E. Development. 1990; 109:635–646. [PubMed: 2401216]4. Mazeyrat S, et al. Nat Genet. 2001; 29:49–53. [PubMed: 11528390]5. Shpargel KB, Sengoku T, Yokoyama S, Magnuson T. PLoS Genet. 2012; 8:e1002964. [PubMed:23028370]6. Rohozinski J, Agoulnik AI, Boettger-Tong HL, Bishop CE. Genesis. 2002; 32:1–7. [PubMed:11835668]7. Pirottin D, et al. Proc Natl Acad Sci U S A. 2005; 102:6413–6418. [PubMed: 15851664]8. Miller JC, et al. Nat Biotechnol. 2011; 29:143–148. [PubMed: 21179091]9. Cermak T, et al. Nucleic Acids Res. 2011; 39:e82. [PubMed: 21493687]10. Guschin DY, et al. Methods Mol Biol. 2010; 649:247–256. [PubMed: 20680839]11. Washburn LL, Albrecht KH, Eicher EM. Genetics. 2001; 158:1675–1681. [PubMed: 11514455]12. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R. Nature. 1991; 351:117–121.[PubMed: 2030730]13. Moscou MJ, Bogdanove AJ. Science. 2009; 326:1501. [PubMed: 19933106]14. Mayer A, Mosler G, Just W, Pilgrim C, Reisert I. Neurogenetics. 2000; 3:25–30. [PubMed:11085593]15. Welstead GG, et al. Proc Natl Acad Sci U S A. 2012; 109:13004–13009. [PubMed: 22826230]Wang et al. Page 4Nat Biotechnol. Author manuscript; available in PMC 2013 December 01.HHMI Author ManuscriptHHMI Author ManuscriptHHMI Author ManuscriptFigure 1. Genetic modification of Sry using TALENs(A) Schematic of Sry TALEN pair 2 and its recognition sequence in the high mobility group(HMG) domain of Sry. TAL repeats are color-coded to represent each of four repeat variabledi-residues (RVDs); each RVD recognizes one corresponding DNA base (NI = A, NG = T,HD = C, NN = G). Nucleotides bound by TALENs are capitalized. Shown below are clones(targeted mutation [TM] alleles 1-3) with Sry deletions induced by TALENs indicated usingdashed lines. Srytm4 (540-bp deletion) and Srytm5 (440-bp deletion) clones are not shown.(B) Southern analysis of targeted alleles. AflII/BsaJI-digested genomic DNA was hybridizedwith a 3’ probe. Expected fragment size: WT = 1.7 kb WT, TM = 3.2 kb. (C) E14.5 controlembryo derived from parental V6.5 mESCs developed as ananatomic male, with testes (t),whereas Srytm1 embryo developed as an anatomic female, with uterus and ovaries (o). b,bladder. Section of control gonad shows Sertoli cell marker expression (SOX9) andtesticular cord formation, while section of Srytm1 gonad shows granulosa cell markerexpression (FOXL2). PECAM marks both endothelial and germ cells. Scale bar unit: · m.(D) Srytm1-bearing offspring of Srytm1 females also exhibited female external genitalia andmammary glands (lower left). Anatomic sex reversal of Srytm1 mice was rescued by the Srytransgene (lower right). (E) Schematic overview of strategy to generate Sry-GFP knock-inalleles. (F) Southern analysis of knock-in alleles. Afl II-digested genomic DNA washybridized with 3’ probe or internal GFP probe. Expected fragment size: WT = 3.9 kb,TM(GFP) = 7.0 kb. (G) RT-PCR analysis of GFP transcript expression in tissues from E12.0Sry-GFP embryos. Internal controls without RT confirmed the absence of genomic DNAcontamination. Gapdh was used as reference.Wang et al. Page 5Nat Biotechnol. Author manuscript; available in PMC 2013 December 01.HHMI Author ManuscriptHHMI Author ManuscriptHHMI Author ManuscriptFigure 2. Genetic modification of Uty using TALENs(A) Schematic overview of targeting strategy. (B) Southern analysis of AvrII-digestedgenomic DNA with 5’ probe or 3’ probe. Expected fragment size: WT , 4.7 kb ( 5’ probe)and 7.7 kb (3’ probe); TM = 12.5 kb for both probes. (C) Schematic overview of strategy togenerate Uty-GFP knock-in alleles. (D) Southern analysis of BamHI-digested genomic DNAwith 5’ probe, 3’ probe or internal GFP probe. Expected fragment size: WT = 10.4 kb,TM(GFP) = 13.5 kb. (E) Bright field/GFP overlay image shows that Uty-GFP mESCsexpressed fluorescent protein. (F) FACS analysis for GFP fluorescence in Uty-GFP mESCsand MEFs, compared with wild-type counterparts. (G) Quantitative analysis of Uty-GFP andUty transcript levels in cells by RT-PCR using primers spanning exon 1-GFP (left) andexons 13-14 (right), respectively. Ki = knock-in.Wang et al. Page 6Nat Biotechnol. Author manuscript; available in PMC 2013 December 01.HHMI Author ManuscriptHHMI Author ManuscriptHHMI Author Manuscript

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TALEN-mediated editing of the mouse Y chromosome

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