| MT is a cysteine-rich, low molecular weight and non-aromatic amino acid protein,andhas a wide range of applications in the field of business and medicine. The traditionalpreparation method of MT has, however restricted its applications,we set out to resolve thepromble with mammary gland bioreactor technology.First, we use the flanking sequence of the bovine αs1-casein gene,-1660-1437,15628-19476, as regulatory elements of MT. The5’ flanking sequence of chickenlysozyme gene and two copies of the chicken β-globin protein insulators were used toeliminate position effects. Through the combination of the three structures, we obtained threeexpression vector pIMC-MT, pIC-MT, pMC-MT.Secondly, the expression vectors were digested by restriction enzyme and thefragement contain the structure elements were used to generate transgenic mice by pronuclearinjection. F0generation mice were screened by PCR and Southern Blot. The pIMC-MT hadseven positive mice (male mice3,14,16,20, female mice22,24,29),and the pIC-MT hadseven positive mice (male mice2,7,8,10,13, female mice21,27),while pMC-MT had threepositive mice (male mice6,12, female mice20). In order to propagate the number of postivefamale mice, F0postive mice were mated to WT mice. Eight postive female mice wereobtained for PIMC-MT (8,10,11,20,22,23,36,44), three for pIC-MT (6,10,28), and onlyone for pMC-MT (25).We finally, collected the milk from F0and F1generation, and breast tissue from F1generation. The expression of MT was examined by Western Blot, RT-PCR and Real-timePCR. We could not detect the expression of MT at the protein level. Transcription of MT wasdetected in F1generation of pIMC-MT-F1-11, and all mice of pIC-MT and pMC-MT.Compared to GAPDH, the level of MT transcription was17.87-fold less in pIMC-MT-F1-11,2.25-fold less in pIC-MT-F1-6,4.92-fold less in pIC-MT-F1-10,14.32-fold less inpIC-MT-F1-28. Only in pMC-MT-F1-25,MT was expressed25-fold higher than GAPDH. |
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