Construction Of Zfn Vectors Targetting β-Lactoglobulin And Exploration Of Zfn Screening Systems

β-lactoglobulin is the most abundant component of the lactalbumin in ruminants. To date, many exogenous proteins driven by the regulatory region of the β-lactoglobulin gene have been specifically expressed in mammary gland. However, β-lactoglobulin is a major allergen to human being. As an effective and specific genome modification technique of comprehensive applicability, ZFN has been successfully used in gene knockout and site-directed recombination in a variety of cells and organisms. It is theoretically feasible to knockout β-lactoglobulin gene via ZFN and insert the exogenous gene into the downstream of the regulatory region of the β-lactoglobulin gene through homologous recombination. This strategy can eliminate allergens in goat milk and achieve the high-level expression of exogenous gene. For this purpose, effective and specific design and screening of ZFN are vital. In addition, a sensitive and stable screening system is very important.In this study, a ZFN basic expression vector harboring RFP as the marker gene was constructed. Thereafter six pairs of ZFN targeting β-lactoglobulin were constructed and different screening methods of ZFN were tested. The result showed that ZFN screening system carrying fluorescent markers was stable and sensitive and then used as the screening system for ZFN in eukaryotes. ZFN pRVNFw-728was found to effectively excise the target gene, which was useful in the knockout of β-lactoglobulin and the effective integration of exogenous genes at the corresponding locus.1. Construction of ZFN basic expression vector. Eukaryotic expression vectors, N13AX·pTARGET and PsvRed·CMV, were constructed. Neo and EGFP gene were marker genes of the former, and RFP gene a marker gene of the latter, all driven by the SV40promoter. Both vectors could be expressed in eukaryotic cells driven by CMV promoter. After transfecting these two eukaryotic expression vectors into the dairy goat fetal fibroblasts cells (gFFCs), the expression of marker genes were observed, indicating the successful construction of vectors. To construct basic ZFN expression vector, NLS and wild-type or optimized FokI genes were inserted into the downstream of CMV promoter in PsvRed· CMV, respectively. The resultant vectors PRVNF and PRVNFw were used for the subsequent ZFN construction.2. Construction and expression of ZFN expression vector targeting goat β-Iactoglobulin. Three pairs of zinc finger protein targeting goat β-lactoglobulin gene were designed. The DNA sequences were synthesized and inserted into two basic ZFN expression vectors PRVNF and PRVNFw. The resultant six pairs of ZFN were transfected into the gFFCs. ZFN expression were detected in the mRNA level.3. Exploration of ZFN screening system. Three ZFN screening systems were tested. Firstly, we constructed the screening system for ZFN carrying fluorescent marker. We inserted the ZFN target sequence into5’end of the green fluorescent protein sequence (the start codon removed) in the pEGFP-N1vector. Green fluorescence was not observed in Hela cells transfected with this vector; and there was probably1/3cells to emit green fluorescence through non-homologous end-joining repaired DSB. After six pairs ZFN vectors were transfected into Hela cells containing target sequence, green fluorescence could be observed in Hela cells of ZFN pRVNFw-728. This result demonstrated that pRVNFw-728could cut the target sequence; moreover, this screening system was sensitive and could be used for the ZFN screening. To improve the detection sensitivity, we constructed the two-fluorescent-markers vector BLG-REG. The BLG coding region and the red fluorescent protein sequence (including the terminator) were fused to the same ORF, then fused to5’end of green fluorescent protein sequence (not including the start codon) in the pEGFP-N1vector, but these two fluorescent marker genes were in different ORF. Goat fetal fibroblast cells transfected by this vector could emit red fluorescence without green fluorescence. When transfecting effective ZFN into Hela cells carrying BLG-REG vector, there was probably1/3cells to emit green fluorescence without red fluorescence, and1/3cells to emit neither green nor red fluorescence through non-homologous end-joining repaired DSB. Secondly, three yeast one-hybrid Bait vectors and six yeast one-hybrid Prey vectors were constructed. In addition, the method of cotransformation using two vectors was optimized. The results showed that200ng of each plasmid,10μL Carrier DNA,100μL yeast competent cells and70μL DMSO resulted in the highest transfection efficiency,6500cfu/μg DNA. Thirdly, PCR and polyacrylamide gel electrophoresis were used to detect the efficiency of ZFN. This method was shown to be unsuitable for fetal fibroblasts cells as a result of low sensitivity.