Establishing Ndufs4 Knockout And STRA8-eGFP Transgenic Mice Using CRISPR/Cas9 Gene Editing Technology

Animal models are one of the tools often used in the field of life sciences.Among them,mice have a variety of CRE tools and can easily achieve conditional knockouts.In various models,mice are favored by researchers.In recent years,with the rapid development of gene editing technology,gene editing technology has been applied to various cell lines,and animals are in full swing.One of the most popular of all is the CRISPR/Cas9(Regular clustered short palindrome repetition/CRISPR-associated protein 9)system.It has the advantages of simple design and high efficiency.Therefore,using this system to obtain transgenic animals has become a trend.The following research has been carried out in this paper:First,we obtained Ndufs4 knockout mice using the CRISPR/Cas9 system.Previous reports have shown that Ndufs4 gene mutations were found in many cases of LS clinical patients.In our study,it was found that knockout mice have significantly lower motor ability and exercise capacity than wild-type mice.This phenotype is similar to LS patients.We further found that knockout mice begin to experience epilation at 3 weeks,and were lighter than wild-type mice.Most mice did not survive adulthood.The surviving mice had low reproductive capacities and could not naturally mating to obtain offspring.Subsequent excision Staining revealed that all oocytes were visible in the ovaries of the knockout mice,and mature spermatozoa could be seen in the testes.Later,we used intracytoplasmic sperm injection(ICSI)to find pure and knockout mice.The offspring are hindered during early embryonic development.Second,we attempted to obtain STRA8-eGFP transgenic mice using both the PITChs system and the HMEJ system.STRA8 is considered to be the marker protein that initiates meiosis.When spermatogonia begin to enter meiosis,Stra8 gene initiates expression,and then the expression is decreased.Therefore,the establishment of STRA8 fluorescence reporter system for mouse reproductive development research has significant meaning for reuse.In this paper,we can use the PITChs method to get the knockout embryos,but then most of the development is arrested in the blastocyst stage.At present,only two surviving mice have been obtained,and the gene sequencing analysis has found that all of them are off-target;and the HMEJ method is used to obtain a comparison in the blastocyst stage.High integration efficiency,and better embryo development,is currently under further investigation.In summary,the present study mainly used CRISPR/Cas9 technology to obtain transgenic mice related to reproductive development research,successfully obtained Ndufs4 knockout mice and STRA8-eGFP knock-in embryos,and will have certain research on future germ cell development.