Establishing NSun2 Gene Knock-out Embryonic Stem Cell Lines And Mouse Model And Researching Gene Function

As is known to all, mammals are close to human on the evolutionary tree, thus making them ideal models for research of gene function and study of pathogenesis. Mice and rats of rodent, pigs, and even non-human primate monkeys are commonly used as mammalian disease models for exploration of gene function, development of new drugs and therapeutic approaches, and study of disease pathogenesis.Embryonic stem cells (ESCs) are derived from the inner cell mass of the pre-implanted blastocysts, and are capable of proliferating unlimitedly and differentiating into all different types of functional somatic cells of the organism, including germline. Due to their capacity of self-renewal and pluripotency, we can easily modify genomic DNA of ESCs and the genetic modification in ESCs can be passed to the next generation through germline. So ESCs are widely used to study gene functions and gain disease models.In order to obtain the genetically modified mammalian disease model, we need to combine the following two aspects together:Firstly, we must carry out genetic modification of genomic DNA, by technologies of plasmid and virus-mediated random integration, transposon system mediated gene trapping, homologous recombination and site specific endonuclease-mediated gene editing. Secondly, we seek to expand the genetic modification to individual level and make the modification transfer to the offspring through germline. Technologies of pronuclear injection, somatic cell nuclear transfer, and embryonic stem cell mediated germline chimeric and intracytoplasmic haploid embryonic stem cells injection into oocytes are used to obtain genetically modified individuals.The site specific endonuclease-mediated gene editing techniques, which is extremely prevalent recently, can modify the genome at specific genomic sites. They are key technologies to interrogate gene function. The most commonly used site specific endonucleases includ Zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly in terspaced short palindromic repeats (CRISPR) system. Especially, CRISPR-Cas9 is widely used for establishment of genetic modification of stem cell lines and mammalian disease models due to its convenience, high editing efficiency and low cost. The CRISPR-Ca9 system consists of a small guide RNA (sgRNA) which target specific genomic loci by complementary base pairing with its homologous sequence, and an endonuclease, Cas9, to generate double strand break (DSB) at the targeted loci by binding to gRNA. The artificial DSBs then initiates the endogenous repairing mechanisms of the cell, including homologous directed repair (HDR) and non-homologous end-joining (NHEJ). As a result, we achieve the insertion or deletion at the specific site by CRISPR-Cas9. What’s more, we also can realize the gene point mutation or even large fragment deletion of chromosome and rearrangement by this technology.As is reported, NSun2 is a RNA methyltransferase that methylates tRNA at the fifth C of cytosine, thus promoting the stability of the tRNA and protein biosynthesis. NSun2 can also reduce Myc induced tumor proliferation and plays a very important role in spermatogenesis. NSun2 gene knockout mice manifest autosomal recessive mental retardation. Recent work has also shown that NSun2 gene is not only involved in the methylation of tRNA, but also play vital important roles in the methylation of mRNA. Previous studies have shown that the sixth N of adenosine methylation modification (m6A) of mRNA has a very important role in mRNA splicing, stability, intracellular localization and translation. According to all above, we speculate that the fifth cytosine methylation modification of mRNA (m5C) may also have similar functions, which will help us to further understand the molecular mechanisms and gene function of NSun2. In my research, we established NSun2 gene homozygous knockout mouse ESCs and NSun2 knockout mice by CRISPR-Cas9 technology, thus underlies the subsequent research of gene fimction and molecular mechanism.