TALENs Mediated Site-specific Insertion Of Human ?-lactalbumin Gene Into Goat ?-lactoglobulin Locus

Goat milk and its products of yoghurt, cheese and powder have great significance in human nutrition by feeding more starving and malnourished people in the developing world than cow milk and treating people afflicted with cow milk allergies and gastro-intestinal disorders. Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as ?-lactoglobulin(BLG). When regarding to BLG, which is scarce in human breast milk, accounts for 53.7% of the total whey protein in goat milk and is a primary milk allergen that causes infant hypersusceptibility. Human ?-lactalbumin(h LA), the main whey protein in human milk, plays a vital role in the synthesis of lactose from glucose and galactose in the mammary gland. Since hLA contains a high proportion of the essential amino acids of tryptophan and cysteine, it has many essential functions in the nervous system, for instance, it can increase brain serotonin synthesis and affect various related behaviors, such as appetite, mood, sleep and cognition. Recently, hLA has been reported to be lethal to tumor cells by inducing tumor cell apoptosis and can effectively cure cutaneous warts caused by human papilloma virus. In recent years, the transcriptional activator-like effector nucleases(TALENs) mediated gene targeting technology is widely applied in the production of transgenic animals. In this study, we applied TALEN mediated homologous recombination(HR) to introduce hLA gene into exon 1 of the BLG locus to produce transgenic dairy goat with hypoallergenic and humanized milk. This research can be divided into five parts:1. Firstly, according to the TALENs cutting sites on the BLG gene, gene targeting vector ploxpII-hLA-neo was prepared in our study. It contains the ploxpII backbone vector, a 738 bp 5' arm and a 714 bp 3' arm which are corresponding to the BLG sequence, a exogenous hLA gene and a neomycin resistant gene cassette which was flanked by two Loxp sites of the same orientation.2. To test whether the hLA gene can be activated and secreted into the extracellular under the control of endogenous BLG regulatory elements, we cotransfected the targeting vector ploxpII-hLA-puro and a TALEN pair to the goat mammary epithelial cells(GMECs). After selected the puro resistant clones, RT-PCR and Western blot analysis were performed on the total RNA and supernatant of GMECs with hormone induction, respectively. The results showed that BLG mRNA levlel was down regulated and hLA was detected existing in supernatant.3. We next cotransfected the targeting vector ploxpII-hLA-neo and a TALEN plasmids or mRNAs to goat fetal fibroblasts or goat ear fibroblasts. After the initial G418 selection, a total of 83 gene targeted clones were screened by junction PCR and long-rang PCR through TALEN plasmids mediated gene insertion. We found that TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection; For TALEN mRNAs mediated homologous recombination, a total of 86 gene targeted clones were identified and the positive clones efficiency was 8.4% which was considerably lower than that of TALEN plasmids mediated gene targeting.4. Several reconstructed cloned embryos were used for 5' junction PCR and long-range PCR analysis to rule out mixed colonies(colonies contained non-targeted cells). Finally, Five mono- and bi-allelic clones with normal chromosome numbers and vigorous cell vability were used as donors for somatic cell nuclear transfer. A total of 717 early reconstructed embryos(2-4 cells) were surgically transferred to the the oviducts of 50 recipient goats. A 34% pregnancy rate was detected 60 days later. A total of six cloned offspring were generated, one was BLG bi-allelic targeted goat and the other five were BLG mono-allelic mutation goats.5. To investigate whether the recombinant hLA can be secreted into goat milk, five pubertal BLGhLA/+ goats were crossed with wild-type goat for natural lactation. After parturition, milk whey proteins from BLGhLA/+ goats were analyzed by 15% SDS gel electrophoresis and Coomassie blue staining. 18-KD BLG bands in the milk from targeted goats reduced, whereas the 14-kD band of ?-lactalbumin increased. As shown by western blot analysis, hLA was highly expressed in BLG+/hLA goat milk compared with that of normal goats. As measured with an enzyme-linked immunosorbent assay(ELISA), the hLA concentration for BLGhLA/+ goat milk was highest at 1.2 mg/mL. A milk composition analysis of fat, protein, lactose and milk solids in the milk revealed insignificant differences between transgenic and non-transgenic goats.