TALEN-mediated editing of the mouse Y chromosome

Abstract

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.

Figure 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 variable di-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 using dashed lines. Sry^tm4 (540-bp deletion) and Sry^tm5 (440-bp deletion) clones are not shown. (B) Southern analysis of targeted alleles. AflII/BsaJI-digested genomic DNA was hybridized with a 3’ probe. Expected fragment size: WT = 1.7 kb WT, TM = 3.2 kb. (C) E14.5 control embryo derived from parental V6.5 mESCs developed as ananatomic male, with testes (t), whereas Sry^tm1 embryo developed as an anatomic female, with uterus and ovaries (o). b, bladder. Section of control gonad shows Sertoli cell marker expression (SOX9) and testicular cord formation, while section of Sry^tm1 gonad shows granulosa cell marker expression (FOXL2). PECAM marks both endothelial and germ cells. Scale bar unit: · m. (D) Sry^tm1-bearing offspring of Sry^tm1 females also exhibited female external genitalia and mammary glands (lower left). Anatomic sex reversal of Sry^tm1 mice was rescued by the Sry transgene (lower right). (E) Schematic overview of strategy to generate Sry-GFP knock-in alleles. (F) Southern analysis of knock-in alleles. Afl II-digested genomic DNA was hybridized 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 E12.0 Sry-GFP embryos. Internal controls without RT confirmed the absence of genomic DNA contamination. Gapdh was used as reference.

Figure 2. Genetic modification of Uty using TALENs

(A) Schematic overview of targeting strategy. (B) Southern analysis of AvrII-digested genomic 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 to generate Uty-GFP knock-in alleles. (D) Southern analysis of BamHI-digested genomic DNA with 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 mESCs expressed fluorescent protein. (F) FACS analysis for GFP fluorescence in Uty-GFP mESCs and MEFs, compared with wild-type counterparts. (G) Quantitative analysis of Uty-GFP and Uty transcript levels in cells by RT-PCR using primers spanning exon 1-GFP (left) and exons 13-14 (right), respectively. Ki = knock-in.