Production of Sry knockout mouse using TALEN via oocyte injection

Abstract

Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.I n most mammalian species, sex is determined by the presence or absence of the Y chromosome. In mice, the Sry gene locates to the minimum sex-determining region of the murine Y chromosome 1 , is expressed in the male genital ridge at the time of sex determination 2 and has been proven to be a sex-determining gene based on gain-of-function experiments, i.e., the overexpression of Sry in XX mice achieved with transgenic mouse technology reveals a male phenotype 3 . Also in humans, the SRY gene has been shown to play a pivotal role in sex determination: point mutations or deletions of the SRY gene are found in approximately 15% of XY females, and translocated SRY is found in the autosomes of most XX males 4 . Although there are a number of suggestive observations, it is important to confirm the function of Sry in vivo using loss-of-function analyses with targeted mutagenesis in order to examine whether Sry is the one and only sex-determining gene on the Y chromosome and to finally confirm the Sry gene as the sex-determining gene and provide an animal model of XY female syndrome. However, it is difficult to create knockout (KO) mice of Y-linked genes using conventional homologous recombination-based methods with embryonic stem (ES) cells, as the process requires an adequate length of specific sequences of homologous arms to construct a KO vector, and the Y chromosome is rich in repeats.In 2013, Sung et al. 5 first reported that KO mice can be produced using transcription activator-like effector nuclease (TALEN) technology without conventional homologous recombination-based methods. TALEN protein is an artificial sequence-specific endonuclease that contains Xanthomonas transcription activator-like effector (TALE) and a nuclease domain of FokI restriction endonuclease 6 . DNA binding domain of TALE consists of a tandem repeat of 33-35 amino acid motifs in which there are two critical adjacent amino acid pairs called a repeat variable diresidue (RVD) that determines the binding specificity for single nucleotide. There is a one-toone relationship between the RVD and its recognition nucleotide 7,8 . Using this code, a TALEN can...

Generation of fertile and fecund F0 XY female mice from XY ES cells

“…Recently, a similarly low fertility and fecundity was reported for XY female mice carrying a 41 base pair (bp) deletion of Sry that was produced by directed targeting with a transcription activator-like effector nuclease (TALEN) (Wang et al 2013). In another example of a TALEN-induced Sry mutation, a single XY female mouse carrying a 2 bp deletion was found to be sterile (Kato et al 2013).…”

Severe myopathy in mice lacking the MEF2/SRF-dependent gene leiomodin-3

“…Lmod3-KO mice were generated using TALEN mutagenesis as previously described (38)(39)(40)(41). The Golden Gate TALEN Kit 2.0 was used to assemble 2 different TALEN pairs targeting the Lmod3 locus, which were designed using the TAL Effector Nucleotide Targeter 2.0 (42,43).…”

Biology of the Sertoli Cell in the Fetal, Pubertal, and Adult Mammalian Testis

“…9.2). Aberrant Sry expression or regulation, however, impedes testis development in XY embryos, whereas a loss of or a mutation within Sry results in ovary development (Battiloro et al 1997;Hawkins et al 1992;Kato et al 2013;McElreavey et al 1996;Nagamine et al 1999;reviewed in Larney et al 2014;Ostrer 2014). Most mutations within Sry disrupt its ability to bind to a consensusbinding motif (A/TA/TCAAA/TG) and bend DNA into its proper conformation (Gubbay et al 1990;Mitchell and Harley 2002;Schmitt-Ney et al 1995).…”