Construction method and application of tmem30a gene mouse model of islet beta cell conditional knockout
A technology of mouse model and construction method, applied in the field of medical engineering, can solve problems such as death, understanding of the pathogenesis of diabetes, and disability
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Embodiment 1
[0042] Example 1. Obtainment of Tmem30a heterozygous mice
[0043] 1) After linearizing the targeting vector Tmem30a tm1a (KOMP) Wtsi (purchased from the Children's Hospital Oakland Research Institute in the United States), the mouse embryonic stem cells 129Sv were transfected by electroporation, the embryonic stem cells were expanded and cultured, and 500 clones were screened, and two strains containing Embryonic stem cells G6 and A11 with the correct target sequence.
[0044] Tmem30a targeting vector Tmem30a tm1a (KOMP) Wtsi structure as figure 1 As shown, the long arm at the 5'end is 4201 bp, and the long arm at the 3'end is 5123 bp; an En2 splicing accepting site (SA) is placed in the second intron, and IRES is followed by the LacZ gene coding sequence, ployA Sequence (PA); after the loxP site is the human βactin promoter and the neomysin coding sequence (neo) to facilitate drug screening; there are two FRT sites at both ends to facilitate the use of FLP tools to delete the rep...
Embodiment 2
[0053] Example 2. Homozygous Tmem30a knock-out mice die in embryonic stage 9.5-12.5 days
[0054] C57BL / 6 / 129Sv heterozygous background mice were selected as experimental mice.
[0055] The Tmem30a KO heterozygous mice obtained in Example 1 were bred with C57BL / 6J mice, and the C57BL / 6 / 129Sv heterozygous background Tmem30a KO heterozygous mice obtained can be born normally and conform to Mendelian laws. There was no significant difference between Tmem30a KO heterozygous mice and wild-type mice. We performed PCR and other methods to detect the offspring produced by mating between Tmem30a KO heterozygous mice, see the results Figure 5 No surviving Tmem30a KO homozygous mice were born. Then we counted its offspring, and the proportions of wild-type and heterozygous were 1 / 3 and 2 / 3 respectively (Table 1). This result is consistent with the Mendelian inheritance of homozygous embryos after death.
[0056] Table 1. Statistical analysis of the offspring of mating between Tmem30a KO het...
Embodiment 3
[0059] Example 3. Construction of Tmem30a conditional knockout mice
[0060] The lethality of homozygous Tmem30a KO affected the in-depth study of its function. In order to study the in vivo function of Tmem30a in various tissues in detail, it is necessary to establish Tmem30a conditional knockout mice.
[0061] The Tmem30a KO heterozygote was mated with FLP deleter (introduced by Jackson Laboratory, USA, strain name B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym / RainJ, also known as FLPer) mice, and the offspring produced two FRTs in the genome The En2-IRES-LacZ-hACT-Neo sequence in between will be deleted, leaving only the loxP sites at both ends of the third exon (see Image 6 ). This animal model is a conditional knockout model of Tmem30a, named Tmem30aTm1.1Xzhu, abbreviated as Tmem30a loxp. Tmem30a loxp / + heterozygote mating with C7BL / 6J can expand the heterozygous population size. Tmem30a loxp / + heterozygous mating, you can get homozygous Tmem30a loxp / loxp.
[0062] Figure 7 The resul...
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