PubMed2001
Human leptin expressed by E. coli had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary glands of transgenic animas. In this study, human leptin gene about 1.0 kb, the terminator of rabbit whey acid protein gene (rWAP) about 0.2 kb and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3 kb were used to construct a expression vector. Before we did the subclonings, the sequences of the human leptin gene were sequenced by ABI377 DNA Sequencer, the results showed that the fragment of human leptin gene included the last nine base pairs of the first exon, the complete sequences of the second exon(172 bp) and parts of the third exon(including part of the encoding sequences and part of the 3' untranslated region). The final expression vector was digested with NotI and a fragment of 7.5 kb was collected and dissolved in TE(10 mmol/L Tris.Cl, pH7.4; 0.1 mmol/L EDTA) for later microinjection. The concentration of DNA was about 2 micrograms/mL, the copy number in 1 mL was about 2.4 x 10(11), every 1 to 2 pL of the prepared DNA solution was microinjected into the mouse embryos at pronucleus stage. After standard microinjection procedures, 48 live mice were obtained. The tails of the mice were cut(about 0.1 g) at four weeks of age, genomic DNA was extracted and digested completely with EcoRI, two were confirmed to be transgenic mice(both were female) by Southern hybridization using DIG labeled human leptin gene as probe, transgenic rate among the mice born was about 4% (2/48). The two female transgenic mice(2# and C3) were mated with nontransgenic male mice. The two founder transgenic mice were segregated with their baby mice for at least three hours at the fifth day after parturition and were milked by intraperitoneal injection of 0.3 IU of oxytocin and udder massage. SDS-PAGE was used to analyze whether there were expression of human leptin in the milk of the two founder transgenic mice with the milk of non-transgenic mouse at fifth day after parturition as control. SDS-PAGE results showed that compared with the control there was a new band in both of the founder transgenic mice milk, and its molecular weight was about 16 kD, which was quite similar with that of the human leptin. The researchers estimated that the expression level of this protein in the milk of the transgenic mice was about 1-2 mg/mL.