earticle

영어

The production of pharmaceutical proteins by transgenic animals is one of the major successes of biotechnology. Knock-in system is a more powerful method to produce mammary gland bioreactor. To date, zinc-finger nuclease(ZFNs), transcription activator- like effector nuclease(TALENs), and clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 systems have been developed for gene targeting. The objective of this study was to develop a knock-in embryo for expression of bovine lactoferrin in the bovine β-casein gene locus by microinjection of knock-in vector with TALEN or CRISPR/Cas9 into bovine zygote. The three kinds of replacement knock-in vectors containing a different length of homologous arm were constructed. These targeting vectors were used enhanced green fluorescent protein(eGFP) as a positive selection marker. These knock-in vectors with TALEN or CRISPR/Cas9 were microinjected into the pronuclear bovine embryo treated cytochalasin B. And the embryos were cultured to blastocyst in the culture medium. These blastocysts were analyzed by PCR to confirm gene targeting by homologous recombination. As a result, when bLF_1kbHR_GFP knock-in vector with TALEN was microinjected into cytoplasm of bovine zygotes, the efficiency of gene targeting was 11.1-14.3%. Also, when bLF_1kbHR and 40HR_GFP knock-in vector with CRISPR/Cas9 was microinjected into cytoplasm of bovine zygotes, the efficiency of gene targeting was 22.2-26.7% but gene targeting by homologous recombination was not detected when bLF_100HR_GFP knock-in vector was microinjected into cytoplasm of bovine zygotes. The precise bovine lactoferrin gene integration of knock-in embryos was confirmed by DNA sequencing analysis. Our knock-in system may help to create transgenic dairy cattle expressing enhanced bovine lactoferrin protein in the mammary gland via the endogenous expression system of the bovine β-casein gene.