Studies On Construction Of Transgenic Vectors Of Human Alpha Lactalbumin And Modified Dairy Goat Fibroblast Cell Lines

Alpha lactalbumin(α-LA)is one of iron-binding whey proteins,which can promote immunity,make lethal to tumor cells and have anti-microbial function,α-LA is rich in essential amino acids and chain amino acids that are useful for baby neurodevelopment,and has been widely used in the infant formula.α-Lactalbumin is the major protein in human milk,about 4.8g/L.Dairy goats,shorter gestation,are smaller size ruminants.Goat milk has higher lactalbumin and milk fat,but α-LA has lower ratio of goat milk.Transgenic technology offers the method to express human alpha lactalbumin and realize humanlized milk,improve quality even yield.Con-struction of transgenic vector is one of the key factors among exogenous gene efficient expression.As reported β-lactoglobulin(βLG)could promote gene to express efficiently in mammary gland,also studies said that chimeric promoters could enhance expression level.At the same time,because gene-targeting could conquer gene silencing resulting from"locus effect"following random integration and make express,it is widely studied.Therefore,we selected hα-LA genome,as the targeted object.Firstly,hα-LA genome sequence including intron,exon 1 to 4,part of 3’flank regulation sequence was cloned and different gene promoters of double marking expression vectors were constructed.Secondly,the effectiveness of these vectors was verified in vitro mammary epithelial cells.Thirdly,these mammary-gland expression vectors were transfected into dairy goat fetal fibroblast cells,and stable transgenic cells integrated with hα-LA gene were obtained by selection.In order to remove the position effect of random integration,gene-replacement targeting vectors concluding double selecting markers of Neo and HSV-TK were constructed,also,were transfected into dairy goat fetal fibroblast cells,screening for gene-targeted modified cell lines,preparation for nuclear cells.The study was divided into four parts:1.The construction of alpha lactalbumin gene specific gland expression vectors and their expression in mammary epithelial cells(GMEC)5.5kb 5’ goat βLG regulation region sequence as promoter,hα-LA genome sequence including intron,exon 1 to 4 and part of 3’ flank regulation sequence as aimed gene,Neo-IRES-EGFP co-expression sequences as selection marker gene,1.7 kb 3’βLG as 3’ flank regulation region sequence,expression elements and expression cassette were spliced together in turn and vector 5A at the length of 15.9kb was constructed.At the downstream of the βLG promoter inserting a CMV enhancer,and B9,a βLG/CMV chimeric promoters expression vector,was constructed,which gave a total length of 16.6kb,Through enzyme digestion,PCR,sequencing,illustrate the validity of these vectors.The lined vectors DNA by Clal were transfected into the dairy goat mammary gland epithelium cells by LipofectamineTMLTX and PLUSTM Reagent.After selection with G418 for about 9 days,G418-risistant green clones were obtained.The transgenic cells were cultured in induction medium containing serum-free DMEM-F12 with prolactin,insulin and hydrocortisone medium capable of inducing recombinant hα-LA expression.Western-blotting analysis demonstrated that the construct-ed mammary gland specific vector 5A and B9 possessed the desirable bioactivity to efficiently express hα-LA in the mammary gland cells and secrete the protein into outside of the cells.At the same time,Paramount to everything,this study laid a firm foundation for preparing the hα-LA gene transgenic goat fetal-derived fibroblast cells.2.Separating dairy goat fetal fibroblasts(gFFCs)and optimizing the transfection system of gFFCs using liposome In order to prepare donor cells for dairy goat transgenic cloning,goat fetal fibroblasts cells(gFFCs)were isolated by attaching tissue explants from a day 30 goat fetus and purified by trypsin.Growth curve showed that the seperating gFFCs were growing well,and eligible for monolayer growth characterization;examined by sex-determined region Y gene(SRY),the female gFFCs were obtained.For a method of foreign gene efficiently transfecting into goats fetal fibroblast cells(gFFCs),using GFP as a reporter gene,transfection condition was investigated and optimized co-mediated by LipofectaminTMLTX and PLUSTM Reagent.Transfection efficiency was evaluated by the percentage of transfected cells with green fluorescence under fluorescence microscope after transfected twenty-four hours.The results showed that gFFCs cultured in 24 well with 6×104 cells/well,transfected twenty-four hours later,0.6μg plasmid DNA per well,ratio of liposome and plasmid DNA 4.5:1 for exposure 6 hours of the cell to DNA-liposome complexes resulted in the highest transfection efficiency,which was 81.2%.3.Establishing gFFCs stablely integrated with human a-lactalbumin The dairy goat fetal fibroblasts were transfected with linearized plasmid 5A and B9 using liposome by the optimized procedure.With the Neo and EGFP gene,selected with G418 for 13-15d,121 and 102 cell clones expressing the green fluorescent protein were separately acquired.Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates.The expanded clones were identified by Nested-PCR,the results indicated that the transgene was stably integrated into the open region of the chromatin of G418 resistant green fibroblast cells.Compared to reporter gene EGFP expression,5A is lighter than B9,and the transgene did not result in the abnormal lities of cell growth,and after frozen 5 months,the cells grow vigorous and their fluorescence was as frozen before.The above results indicated that these transgenic cell clones can be competent as donor cells for creating a transgenic goat by SCNT.4.Constructing of TK negative selection marker gene targeting vector and selection of site-specific cells we subcloned two single directional HSV-TK genes into the two sides of homologous arm of express vectors 5 A and B9,which were constructed in part 1,as a negative selection marker gene,5’βLG as the 5’ homologous arm,3’βLG as the 3’homologous arm,construct targeting vector TK-5A and TK-B9 at the lotus inβ-lactoglobulin gene of the protein genes.The recombinant plasmids were identified by size,restriction fragment analysis and partial DNA sequencing.The results illustrated that the structure of the finally constructed vector accords with the designed plasmid map.Vectors were digested by XhoI to remove prokaryotic sequence and then transfected into gFFCs,selected with 1000μg/mL G418 and 6μM GANC.17 TK-B9 green resistant clones and 28 TK-5 A ones were aquired to be identified.