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以含有 1.6 kb的小鼠乳清酸蛋白 (WAP)上游调序列的 p CAT- WAP和含有人 c- myc c DNA的 p WM为原始质粒 ,构建了 WAP启动子调控下的 c- myc乳腺定位表达载体 p WCS。载体用 Bgl +Bam H 酶切 ,回收 3.5 kb的基因片段 WAP- c- myc- SV40 Poly A,通过显微注射方法导入 C5 7BL/ 6 J× DBA/ 2 JF1 代小鼠受精卵的雄原核内。共注射10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 45只。PCR检测 ,阳性鼠 9只 ,South-ern blot检测 ,阳性鼠 3只 ,其中 1只母鼠 ,2只公鼠 ,在饲养过程中 ,1只母鼠意外死亡。对 2只转基因公鼠的 F1代PCR检测表明 ,仅 1只公鼠具有遗传性 ,在所生的 2 7只 F1代小鼠中有 13只为 PCR阳性。
The c-myc breast localization under the control of the WAP promoter was constructed by using p CAT-WAP containing the upstream sequence of 1.6 kb mouse whey protein (WAP) and p WM containing human c-myc c DNA Expression vector p WCS. The vector was digested with Bgl + Bam H and the 3.5 kb gene fragment, WAP-c-myc-SV40 Poly A, was recovered and introduced into the pronucleus of the fertilized mouse of C5 7BL / 6 J × DBA / 2 JF1 generation by microinjection . A total of 100 eggs were injected, and about 600 surviving eggs were transplanted into the fallopian tubes of 29 females, respectively, 45 of which were offspring. PCR detection, positive 9 rats, South-ern blot test, positive 3 rats, including 1 female and 2 male rats during feeding, one female died accidentally. Fl generation PCR tests on two transgenic males showed that only one male mouse was heritable and 13 of 27 given F1 generation mice were PCR positive.