The Establishment Of A Method Of Gene Insertion At Bovine Î'-casein Locus With HLf Gene Mediated By CRISPR/Cas9

Multiple effects of lactoferrin(Lf) with antibacterial and immune regulation functions had been confirmed, meanwhilehuman lactoferrin(hLf) has been used to reduce the use of antibiotics, prevent infection in infants, strengthen immune system and defeat cancer. It has been used for health food, drug production and many other domains. Realization of transgenic cows secreting human lactoferrin is a very attractive potential human lactoferrin sources, while the genome targeting of somatic cell and somatic cell nuclear transfer technology have provided a strong technical support for the realization of this purpose. Among the recent gene targeting technologies, CRISPR/Cas9 and zinc finger nucleases(ZFNs) Gene Targeting System are parts of the most widely studied, and both have been used successfully in mice, pigs, cattle, human cells, et all. CRISPR/Cas9 Gene Targeting System has not been use for inserting gene in livestock’s genome widely and has a simple-design advantage than ZFNs. The purpose of this study is to build an efficient and reproducible gene targeting method with the use of CRISPR / Cas9 system, which is to insert human lactoferrin gene into the β-casein site(CSN2) of bovine fetal fibroblast(BFFs) genome. The production of transgenic embryos for embryo transplants, which can lay the groundwork of the researches of transgenic cows with human lactoferrin and further production of human lactoferrin. The main contents and results are as follows:1. Construction and activity analysis of CRISPR/Cas9 Gene Targeting SystemCas9 protein sequence of the codon had been optimized and cloned into the pEGFP-C1 vector, to from a new vector named pCas9-C1. The guide-RNA(gRNA) which is designed and synthesized had been cloned into pSilence 2.1-u6 vector to from the pSilence-gRNA vector. The activity analysis of CRISPR/Cas9 system had been accomplished by co-transfection of BFFs with the two vectors, and analyzed the sequence of cleavage site with PCR and CEL-I analysis.2. Site-directed Gene Insertion mediated by CRISPR/Cas9In this study, the targeting vector pCSN2-hLf had been formed by inserting the hLf gene sequence into the multiple cloning site of general targeting vector pTCSN2 constructed by Dr. Xiang Liuxu. The vectors of CRISPR/Cas9 and ZFNs(Constructed by Dr. Liu Xu) co-transfected BFFs separately with vector pCSN2-hLf, and screened through G418 for stably transfected cells. The G418-resistant colonies were transferred to a 96-cell clones to expand cultivation, and was found positive rate of the gene targeting at CSN2 site with CRISPR / Cas9 system was 9.7% and ZFNs 8.6% by PCR analysis.3. Production of transgenic bovine embryos by somatic cell nuclear transfer(SCNT)Selected suitable positive cell clones as transplant donor and collected oocytes as receptor, SCNT was to produce nuclear transfer embryos.2 days after SCNT, the cleavage rate was 84.6%; and after 7 days, the blastocyst rate was 33.3%. After PCR analysis of the positive bovine transgenic embryos of hLf gene can be used for the production of transgenic cloned cows.Combined with the results above, show that CRISPR/Cas9 can insert the hLf into CSN2 site of BFFs genome successfully, and the formated transgenic embryos can be used for the production of transgenic cloned cows; CRISPR/Cas9 has nearly the same positive rate as ZFNs, and the effect of off-target effect needs further validation.