Study On The Construction Of Transgenic Mice And Bcl-2 And C-myc Mammary Expression System

The 1.61kb 5' promoter upstream regulatory sequence of the mouse, whey acid protein gene was amplified by PCR.from the mouse genome.The result showed that the sequence was the same as the published.A Mammary-specific expression vector ,pWTS,was constructed using the regulatory sequence fused with tPA cDNA in order to determine whether the promoter could guide the foreign gene expression in mammary glands. pWTS ,and three other mammary-specific expression vectors,pRWTS, pBTS and pCTS were mixed with lypofectaminein a certain ration,respectively and were injected into the mammary glands at later stage of gestation of pregnant rabbits through teat canal.The result of ELISA assays showed that the above vectors could direct tPA expression in rabbit mammary glands.The expression level of pWTS was the highest.Two mammary-specific expression vectors,pWBS and pWCS,were constructed by fusing 1.61 kb WAP 5' upstream regulatory sequence with 0.9kb human Bcl-2 cDNA and 1.4kb human c-myc cDNA.Both Bcl-2 and c-myc transgenic mice were established by microinjection of the purified 3.0kb Bcl-2 and the 3.5 kb c-myc expression cassettes.Six hundred ova were transfered into twenty-nine pseudopregnant recipients. Fifty-five offspring were born from the ova mircroinjected with the Bcl-2 fragment expression cassette.Twelve of them were proved to be gene integration positive by PCR .Four founder mice,two male and two female ,were further confirmed to be transgenic by Southern hybridization. Two male mice died by accidentduring their feeding.One of the two female mice was proved to express the Bcl-2 gene in mammary gland by Western blotting analysis. Nine female mice among 45 Fl generation mice were proved to be transgenic mice by PCR. No visible tumor on the skin of the mammary gland were observed during five months of mutiple pregnancy and lactation.fourty five offspring were born from the ova mircroinjected with the c-myc fragment expression cassette.Nine of them were gene integration positive by PCR.Three mice, two male and one female ,were further confirmed to be transgenic by Southern hybridization. One female mouse died accidently during its feeding.One of the two male mice could transmit the c-myc gene into its Fl generation which was proved by PCR Six females of thirty five mice in its Fl generation were comfirmed to be transgenic by PCR.They are now under mutiple pregnancy and lactation.The results have provided a model of transgenic mice for further studying the effect of Bcl-2 and c-myc in vivo in the development of breast tunor.