ABSTRACT
Human ALR gene sequence was amplified by PCR from human total DNA and inserted into pIRES(2)-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo(r) and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/ALR by the induction of lipofectAMINE(TM). The positive cell clones were selected with medium containing G418 (800 µg/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and immuno-histochemical staining. Results showed that the EGFP and ALR genes linked with IRES were coexpressed simultaneously in sFFCs; the blastocysts formed by nuclear transfer using tranfected donor cells are all transgenic blastocysts. EGFP, ALR and Neo(r) gene were all expressed in the transgenic embryos. In conclusion that a method to construct the positive embryos before pre-implantation which stably express ALR gene by the indication of EGFP expression has been successfully established. The application of this method can simplify the procedure of testing the targets and contribute to the efficiency increasing of transgenic domestic animal production.
Subject(s)
Animals, Genetically Modified/genetics , Fibroblasts/metabolism , Gene Expression , Proteins/genetics , Sheep/genetics , Transfection , Animals , Animals, Genetically Modified/embryology , Animals, Genetically Modified/metabolism , Cells, Cultured , Embryo, Mammalian , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Nuclear Transfer Techniques , Oocytes/metabolism , Proteins/metabolism , Sheep/embryology , Sheep/metabolismSubject(s)
Cyclin-Dependent Kinase 2/physiology , Meiosis , Oocytes/cytology , Spermatocytes/cytology , Animals , Female , Humans , Male , MiceABSTRACT
Fertilin beta may play an important role in sperm-egg plasma membrane adhesion and fusion. To explore the effects of fertilin beta, the sperm membrane protein subunit, in the sheep fertilization process, we used RACE technique for cloning the full length cDNA of the coding region (CDS) of fertilin beta. The coding region was 2, 217 bp, which consists of 738 amino acids. The fertilin beta in the sperm membrane of sheep shares 79.4%, 66.7%, and 58.1% sequence identity with that in bovine, pig and human, respectively. The phylogenetic analysis of the fertilin beta gene family indicated the fertilin beta was clustered with bovine, and is closest to the one of bovine. This result is consistent with the result of the traditional classification. The protein structure analysis showed the disintegrin domain of sheep fertilin beta contains a TDE. Besides the above tripeptide sequence, the family member of ADAM (A Disintegrin and A Metalloprotease) follow the conserved sequence of ECD of X-D/E-E, and formed the conserved sequence of X-D/E-ECD. The pentapeptide sequence of the sheep fertilin beta is TDECE.
