| Lipoprotein lipase (LPL) is an important enzyme in the lipid metabolism, which catalyzes the hydrolysis functon of the chylomicrons triacylglycerol component and very low density lipoproteins, thereby providing fatty acids and monoacylglycerol for tissue utilisation. LPL gene mutation may affect the activity of LPL, and result in lipid metabolism disorder. It is associated with typeⅡdiabetes, hypertension, atherosclerosis, obesity and corony artery disease. Gene knockout is a new technology of molecular biology which bases on DNA homologous recombination. And animal models of gene knockout have already been used in many studies. Swine is an excellent animal model of human cardiovascular disease, as it is similar with human in hematology and haemodynamics. So if we modify the LPL gene, we will get the transgenic swine which is useful in research of cardiovascular disease. In our study, LPL gene knockout was constructed with gene targeting technology; porcine fetal fibroblasts were transfected with Lipofectamine TM 2000; then transfected fibroblasts were selected and identified.Two pairs of primers were designed by Oligo 6.0 software and synthesized according to swine LPL mRNA sequence which had sequenced from GenBank. Genomic DNA extracted from the liver of Landrace was obtained as a template. IntronⅡand intronⅢof porcine LPL were amplified by PCR technology, and results show that specified bands were about 7.5kb and 2.1kb of length. Then porcine LPL intronⅡand intronⅢwere connected with pGEM-T vector. Take the pGEM-intronⅡplasmid and pGEM-intronⅢplasmid as template, homologous long arm and short arm of LPL gene knockout porcine vector were amplified, with the length of 4.1kb and 1.3kb. Then the intronⅡand intronⅢwere connected with the pGEM-T vector. The result was homologous compared with porcine LPL intronⅡand intronⅢ, showing that the homologous long arm had 99.7% homology with porcine LPL intronⅡ; and the homologous long arm had 99.9% homology with porcine LPL intronⅢ. Finally, the homologous long arm was digested by Sal I and Xho I; the homologous short arm was digested by Cla I and AflⅡ. The two fragments were inserted into both sides of Neo multiple cloning sites of pSSC-9 vector. Then the pSSC-Larm-Sarm vector of porcine LPL gene knockout was successfully constructed.The minipig fetal fibroblasts were selected as the donor cells in this experimenl, which they could have more times for passage, long time of in vitro culture and also stable karyotype. G418 cell toxicity test of the cell line show that, when concentration of G418 was 350μg/ml, the normal cell died on 7 day. So this concentration was selected for transfection. The cells were transfected with LipofectamineTM2000. The pSSC-Larm-Sarm vector of porcine LPL gene knockout would be transfected into fetal fibroblasts, which based on the principle of homologous recombination. Then the exonⅢof porcine LPL was knocked out. The transfected cells were selected with G418 for 5-7 days, G418 and GANC for about 7-10 days. The dipl-resist cells were collected and identified by PCR. In order to improve the efficiency of PCR amplification, the three pairs of primers were designed: a pair of specific primers in the neo gene and lateral of the short arm, a pair of primers in neo gene and a pair of primers in TK gene. PCR amplified products of positive cells should be 2095bp, 409bp or non-amplified products. After PCR identification we found homologous replacement occurred in three out of selected six cell clones.In short, in this experiment porcine LPL gene knockout vector and LPL gene knockout cell lines were successfully built, which were important in the construction of LPL gene knockout swine.
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