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The production of therapeutic protein and improve of productivity of the domestic animal from transgenic animal is the major technology of biotechnology. Lactoferrin has its highest content in the colostrum, and is known as the antiviral substance. Insulin like growth factor1 is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that insulin like growth factor1 gene is inserted into the β-casein gene locus for expression of bovine insulin like growth factor1 (bIGF1) on the bovine β-casein gene. The knock-in vector consists of 5' arm region (1.02 kb), insulin like growth factor 1 cDNA, CMV-EGFP, and 3' arm region (1.83 kb). To express bIGF1 gene as transgene, the F2A sequence was fused to the 5' terminal of bIGF1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF1 gene. These knock-in vectors may help to create transgenic dairy cattle expressing bovine IGF1 protein in the mammary gland via the expression system of the bovine β-casein gene.
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.