Production Of Gene Targeting Goat By Insertion Of Hlf Gene Into β-lactoglobulin Locus

Goat milk is known as “the king of milk”, but part of people are found to be allergic to the goat milk proteins. β-lactoglobulin(BLG) is considered a major allergen in goat milk. The milk allergy problem has been solved through several biochemical technologies, but all of these methods cannot fully solve this problem. Gene targeting can work as a direct and complete method to reduce and even eliminate the allergen BLG from goat milk without any change in the other milk components. Lactoferrin(LF), an iron-binding glycoprotein of the transferrin family, showed antibacterial, anti-tumour, antiviral, immunomodulatory, and several other biological functions. In this research, ZFN and TALEN-mediated homologous recombination(HR) was used in goat somatic cells to knock out BLG followed by hLF gene knock-in. Consequently, gene targeting goats were produced. Following this, the allergen of goat milk can be reduced without changing the balance of milk composition. Moreover, the pharmaceutical protein hLF may also be produced in the mammary glands through the BLG promoter. These results will facilitate the improvement of the quality of goat milk products.1.gene targeting vectors construction. The donor plasmid pG and pSHQ were constructed to introduce an insertion fragment containing the hLF cDNA-bGH p A and an autonomous expression resistance cassette as positive selection gene flanked by two loxp sites into the BLG loci. The donor plasmid pG contains a neomycin-resistance cassette, while the pSHQ contains a EF1α-GFP-p2A-puromycin selection cassette. After the plasmid pG was transfected into the E.coli BM25.8 which expresses the Cre protein, the positive selection gene flanked by two loxp sites was removed successfully, revealing that the two LoxP sequences in the same direction can be used to remove the positive selection gene of clones.2. The function of gene targeting vectors detection. ZFNs or TALENs’ plasmids were cotransfected with targeting donor pG into dairy goat fetal fibroblasts(GFFs). The junction PCR results showed that ZFN and TALEN both can mediate the hLF gene insertion into the BLG locus. The cells only transfected the donor pG group could not be amplified the anticipative products, demonstrating that the ZFN and TALEN can improve the efficiency of homologous recombination. TALENs’ plasmids were co-delivered with donor pG into goat mammary epithelial cells to verified the expression of hLF. After selection with G418, the G418 resistant cell clones were cultured and induced for lactation, the RT-PCR and western blot results showed that the hLF can be expressed sussfully after homologous reconbination.3.Targeting efficiency of ZFN and TALEN mediated homologous recombination. To detection the efficiency of homologous recombination mediated by ZFN and TALEN, ZFNs or TALENs’ plasmid or mRNA were cotransfected with the donor pG into GFFs, the G418 resistant clones were identified by junction PCR、long-range PCR and sequencing to select the gene targeting positive clones. And then the efficiency of homologous recombination about ZFN and TALEN was compared. The results showed that, 996 G418 resistant-clones were picked, among which 127 were positive, the rate of positive cell clones was 12.8% in TALENs’ plasmid mediated group; 832 G418 resistant-clones were picked, among which 69 were positive, the rate of positive cell clones was 8.3% in ZFNs’ plasmid mediated group; 810 G418 resistant-clones were picked, among which 73 were positive, the rate of positive cell clones was 9.0% in TALENs’ mRNA mediated group; 821 G418 resistant-clones were picked, among which 48 were positive, the rate of positive cell clones was 5.8% in ZFNs’ mRNA mediated group. The positive efficiency of TALEN group was higher than ZFN. The positive efficiency of TALEN or ZFN plasmid transfection group was higher than mRNA group. Considering the random integration of plasmid group, the ZFNs and TALENs’ mRNA transfection groups were selected for Somatic cell nuclear transfer(SCNT).4.Generation of gene targeting dairy goats through SCNT. Karyotype analysis of the gene targeting positive cells revealed that most of the clones have normal karyotype. So the cell clones with normal karyotype and in good shape were used for SCNT. Fusion rate, cleavage rate and blastocyst rate were statistical analysed after SCNT and compared with the negative group. The cell clones without significant difference in fusion rate, cleavage rate and blastocyst rate were used for producing clone embryos and embryo transplantation. The developed cloned embryos were transferred to 37 recipient goats, consequently, four goats were born and alive(three from TALEN-mediated group, one from ZFN). PCR, sequencing and southern blot demonstrated that all four goats were mono-allelic targeted goats. The sequencing results showed that the 2,379-bp hLF cassette was inserted following the signal peptide sequence of BLG gene in BLG+/hLF goats and replaced 17 base of the BLG gene.5.Analysis the reproductive performance and the milk components of gene targeting goats. The reproductive performance of gene targeting goats did not have significant differences. All four transgenic goats could pregnancy and lamb normally. Especially, one of the offspring was also mono-allelic targeted goat, which has the same genotype with female parent, revealing that the targeing gene modification can be transmitted to offsprings.Milk from gene targeting goats was tested after lambing. The SDS-PAGE, western blot and Q-PCR results showed that the hLF protein was expressed in the transgenic goat milk successfully and the BLG mRNA expression levels were decreased, the other protein components have no significant differences with wild-type milk. The fat, total protein, lactose and dry matter in gene-targeted goat milk also have no significant differences with wild-type milk.In conclusion, ZFN and TALEN were applied to mediate homologous recombination in somatic cells and produce gene targeting dairy goats combined with SCNT, improving the transgenic animal cloning technology system using artificial nuclease-mediated homologous recombination. Following this, the transgenic goat milk with the allergen eliminated and without changing the balance of milk composition was produced. Moreover, the pharmaceutical protein hLF was added to the goat milk. Our study laid a solid foundation for the improvement of the nutritional value of goat milk and the cultivation of new goat varieties.