| Animal transgenic research is basically characterized by changing the genetic make-up of animals, although the purposes of changing the genetic make-up of animals are always different. In our study we hoped to donate transgenic animals a new phenotype by changing their genetic materials. The animals used in this study were KunMing White mice.With the improvement of living standards obesity has increasingly been a serious social problem. It is proved by modern medical sciences that obesity is closely related with the incidence of diabetes, heart disease, and hypertension. It is clear that leptin encoded by obese gene is one of the most important regulating factors of fat metabolism and body weight. Whether we can use transgenic animals of obese gene to express human leptin and whether we can use this recombinant protein to treat the diseases related with obesity are the purposes of our doing this study. In this study, three plasmids were used to construct the fusion expression vector. A fragment of human obese gene (cDNA) about 1.0kb was cloned in plasmid pBIuescript, the terminator of rabbit whey acid protein (rWAP) gene about 0.2kb was cloned in the plasmid pR, and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3kb (-6300bp-+28bp) was cloned in the plasmid p26. Before we did the subclonings the sequence of the obese gene was automatically sequenced by ABI377 Sequencer, the sequencing results showed that the fragment of the cDNA of human obese gene included the last nine basepairs of the first exon (21bp-29bp, the whole sequence of the first exon is 29bp), the complete sequence of the second exon (172bp) and parts of the third exon (including the whole encoding sequence and part of the 3' untranslated region). The typical codon ATG for initiation of translation and TGA for stopping translation were included in the encoding sequence (504bp). The final expression vector plasmid p22 was digested with NotI and the fragment of 7.5kb was dissolved into TE (10 mmol/L Tris-Cl, pH 7.4; 0.1 mmol/L EDTA) for later microinjection. The concentration of the fragments was 2ug/ml, the copy number in lml was about 2.4x10", every 1 to 2pl of the prepared DNA solution was injected into the embryos at pronucleus stage . After standard microinjection procedure, 48 live mice were obtained, the tails of the mice were cut at four weeks of age, genomic DNA was extracted. Two pairs of primers were designed according to the rWAP promoter sequence and human obese gene sequence, the sequences of the two pairs of primers for PCR amplification were as follow:P,: 5'-Tgg, AAg, gAC, Agg, ATg, ggg, Tgg, Ag-3' P2: 5'-gAT, AAg, gTC, Agg, ATg, ggg, Tgg, Ag-3' P3: 5'-ggg, ATC, CgT, gCC, gAT, CCA, AAA, AgT, CCA, AgA-3' P4: 5'-CgA, ATT, CCC, TTC, AAg, gCC, TCA, gCA, CCC, Ag-3'It was estimated that P, and P2 could amplify a fragment of 1.1 kb, P3 and P4 could amplify a fragment of 460bp. However the amplification results in this study were not very stable, then genomic DNA was digested completely with EcoRI, two were confirmed to be transgenic mice (both were female) by Southern hybridization using the human obese gene as probe, transgenic mouse rate among themice born was about 4%(2/48). On basis of this we could come to such a conclusion, PCR amplification was low cost and quick for testing, but it was easy to be polluted and caused false-positive results; compared with this Southern blotting was high cost and took long time, but it was very stable and reliable. So we suggested the researchers test transgenic animals directly by Southern blotting. The two female ransgenic mice (2* and C3) were mated with nontransgenic male mice. After full-term pregnancy and parturition 2* and C3 gave birth to 7 and 8 baby mice respectively. Every three of the 7 and 8 born by 2* and C3 were confirmed to have the transgenes in their genome by Southern hybridization with the human obese gene as probe.The results showed that the two founder transgenic mice were normal in reproduction and the transge...
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