Study On The Function Of KIAA1429 In Mouse Embryonic Development

With emerging revolutionized tools employed in life science research in recent years,many new genes are discoverred and dicipherring the funtions of these new genes not only helps us to better undstand how the living organism works,but also leads to development of biomedicals for improvement of human health.KIAA1429 is widely found in animals and it has been found that the gene is involved in methylation of 6-methyladenine of mRNA.But the role of KIAA1429 in this process is unclear.In this study,mice were use as a model animal and CRISPR/CAS9,molecular cloning,micromanipulation,embryo transfer,cell transfection and flow cytometry were included in our toolbox to explore the funtion of KIAA1429 during in embryonic development and cell survival.The results are as follow:Section 1:Effect of mutant KIAA1429 on embryonic stem cells.1.The construction of the vector was carried out by using CRISPR technology.Embryonic stem cells were transfected and flow cytometry was performed to sort the transfected cells.2.Sorted cells were cultured in two format,bulk-sort culture and clonal-sort culture.Sequencing anlysis revealed that cells carried mutant KIAA1429 was found in first passage after bulk sort,but abscent after the third passage.And not frame-shift mutation was found in clonal sorting These results indicate that K1AA1429 is required for viabiligy of embryonic stem cells and the cells carried the mutant copy cease to grow during expansion.Scetion 2:Effect of KIAA1429 on embryonic development.We use bioinformatic tool to analyze the KIAA1429 gene design gRNA to contruct CRISPR/Cas9 targeting plasmed.After injection of the targeting plasmid,embryos were transfer and examine the embryo development.The results showed that no live pups was obtained,suggesting that KIAA1429 plays an important role in embryonic development.2.Next we analyze the KIAA1429 in arrested embryos and blastocysts in vitrofound that embryos with mutant KIAA1429 are still capable of developing to blastocyst stage.3.Anatomical examination of pregnant rats at different time points after embryo transfer demonstrated that the embryos development is blocked six days after transplantation.In summary,these results suggest that KIAA1429 is an important gene associated with embryonic development,and its absence leads to fatality of embryo.