Construction Of Bovine Transgene T-PA Fetal Fibroblast Cell Lines And Its Nuclear Transfer Embryo

In the research, the mammary gland-specific expression vector pBC1-TPA and pBC2-TPA were constructed, which were consisted of regulating elements regulation region of bovineβ-casein and expression sequence finger-domain lacking t-PA cDNA. Based on them, matrix attachment regions in the human serpin gene(α1-AT MAR) were inserted. Therefore, the other two mammary gland-specific expression vector pBCHM1-TPA and pBCHM2-TPA for finger-domain lacking t-PA were constructed. Mouse mammary epithelial cells were transfected by these four vectors, finger-domain lacking t-PA was expressed well, especially in pBCHM2-TPA. Bovine fetal fibroblast was transfected by pBCHM2-TPA, and stable transfected cell clones were obtained and used for nuclear transfer. Finally, transgenetic clone embryoes derived from nuclear transfer can be used for producing the mammary gland bioreactor. The results of this study is as following:1. Eukaryotic DNA element called Matrix Attachment Regions (MARs) can function on regulating the structure and activity of chromosome. In the research, Matrix attachment regions in the human serpin gene(α1-AT MAR) was cloned from human genomic DNA, it was about 1 252 bp. Sequence analysis indicated that it involved some important functional regions, such as AT-Richness Pattern,ORI Pattern,ATC Rule and so on.α1-AT MAR was inserted into pEGFP-C1 and located in the downstream of EGFP, and the recombination expression vector pME was constructed. Then pME and pEGFP-C1 were transfected into human embryonic kidney 293 cells (293T), respectively. After G418 fastness screening, clones were assayed after 20 days of selection of G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulate nearby gene expression. Then, chromatin immunoprecipitation (CHIP) where it co-localizes with newly CMV promoter and RNA polymeraseⅡ(RNAPⅡ) is detected by PCR, The result demonstrates that more RNAPⅡwas recruited to the CMV promoter in presence of MARs.α1-AT MAR could improve gene expression level.2. Three fragments of bovine beta-casein (about 9.7 kb) were cloned by PCR, and then were subcloned into pGEM-T easy Vector. After sequence analysis on NCBI with Blastn, the results indicated that the fragments had the homology of 98.0% respectively with the correspondingly region of bovine beta-casein. And these three fragments includes integrated 5'flanking sequence, eight exons and partial the eighth intron. Based on them, two mammary gland-specific expression vectors were constructed. One of them use fused fragments of CN1 and CN2 as 5'promoter, splicing fragments of CN3 and 600 bp poly(A) by recombinant PCR as 3'promoter. The other used CN1 as 5'promoter, 600 bp poly(A) as 3'promoter. Both of them can be applied in preparing mammary gland bioreactor.3. Using double digestion, the requisite expressing sequence of human finger-domain lacking t-PA cDNA was merged with the commonly primary expression vector pBC1-TPA and pBC2-TPA. Then,α1-AT MAR was inserted into two vectors, and pBCHM1-TPA and pBCHM2-TPA were constucted successfuly, which also included antibiotics gene (kana/neo) and report gene (EGFP). These four vectors were transfected into mouse mammary epithelial cells, the expression of report gene EGFP observed under fluoroscope. Western-blot result indicated that the expression of human finger-domain lacking t-PA was well, especially in pBCHM2-TPA.4. Mammary gland-specific expression vector pBCHM2-TPA was transfected into bovine fetal fibroblast. After stable transfected cell strains were obtained, the expression of TPA was tested by RT-PCR. The results of PCR and Southern-blot shown that human finger-domain lacking t-PA was integrated into bovine genome, which can be used for nuclear transfering.5. We constructed bovine nuclear transfer embryo with transgene (t-PA) fibroblast, during which time the electrofusion parameters were optimized, the reconstructed embryo developmental data were recorded under microscopes and the t-PA gene expression in the embryo was detected as well. The results showed that, compared to normal fibroblast, the fusion could only be accomplished at higher voltage in transgene fibroblast, and the parameters were: 1500 V/cm, 20μs/each time, 1 s interval. The green fluorescence of GFP expressed in reconstructed embryos of over 8-16 cells, and since then, the expression level increased along with the embryo development. RT-PCR also demonstrated the extra gene has constructed into new embryo chromosome, which indicated that it was possible to produce transgene bovine embryo through somatic cells gene transfer.