Preparation Pig Cell Lines Of Transduction AMPD1 Gene

Adenosine monophosphate deaminase(AMPD)is one of the key enzymes in the purine nucleotide cycle.It has only been found in all eukaryote . AMPD is a complex allosteric enzyme encoded by a multigene family in mammals .The genic activate can protect muscle cells from lack of energy. Its function is lost will lead to a metabolic muscle disease .,The study changed expression and function research of AMPD1gene ,in order to produce livestock which have more muscle and more flavor. This study was designed to clone the cDNA of pig boar AMPD1 gene based on the sequences of AMPD1 gene of Genbank. The method by polymerase chain reaction (PCR) to cloned cDNA sequence of AMPD1 gene of pigs .The method by double enzyme cut and overnight connection to complete coding region of AMPD1 gene connected pcDNA3.1 (+) eukaryotic expression vector.fibroblast and pk15 cell were successfully established by conventional method. Design for four different of transfection system, namely, DNA: Lipofectamine = 1:2/1:3/2:5/2:7, Comparison analysis of transfection efficiency afer over the transfection.Construct plasmid was introduced into fetal fibroblast and pk15 cell of pig by lipofectamin-mediated DNA transfection methods, Cells selecting with G418 for 6 days, then go on selecting with G418 for 14 days of cells. At last, the positive cells were identified by PCR. The main results are as follows:1)The complete two thousand two hundred and seven tcDNA sequences of minpig AMPD1 gene were cloned .2)Constructs pcDNA3.1 that AMPD1 gene of pig eukaryotic expression vector after double enzyme.3)Fibroblast of pig selecting with G418(200μg/ml) for pcDNA3.1(+)-AMPD1 plasmid, and PK15 cell of pig selecting with G418(1000μg/ml) for pcDNA3.1(+)-AMPD1 plasmid .After transfection, ,then fibroblast of pig and pk15 cell of pig go on select with 100μg/ml G418 and 500μg/mlG418. DNA and Lipofectamin than for 2:5, stable transfection 6 hours are optimized condition for transfection in two kinds of cells.4)Two cells that stable expression AMPD1gene all have purpose gene amplification,it shows that transfection success.5)Get stable integration AMPD1 gene of pk15 monoclonal cell.