【摘要】 构建小鼠嗜铬颗粒蛋白Ａ（Ｃｈｒｏｍｏｇｒａｎｉｎ Ａ，ＣＧＡ）基因的反义ＤＮＡ载体ｐＧＡＳ１Ｃ－ｌａｃＺ。 用电穿孔的方法将该载体转化大鼠肾上腺髓质细胞瘤细胞系ＰＣ－１２，Ｘ－Ｇａｌ染色后证明位于 ＣＧＡ基因启动子下游的报告基因ｌａｃＺ已经表达。用限制性内切酶除去载体的质粒骨架后，显 微注射入供体昆明小鼠的受精卵中，随后将注射过ＤＮＡ的受精卵移植入假母的输卵管中，完 成正常的胚胎发育。用ＰＣＲ的方法筛选假母产下的小鼠，得到ＣＧＡ基因反义ＲＮＡ转基因首 建鼠１４只。将首建鼠分别与正常昆明鼠交配，产生后代。取首建鼠的肾上腺进行Ｘ－Ｇａｌ染色， 组织用于石蜡切片，根据各鼠肾上腺切片的蓝色深浅判定转入基因表达量的高低，筛选到两 只表达量高的首建鼠，留下它们的后代。取转基因鼠的各种组织用于Ｘ－Ｇａｌ染色，发现报告基 因在肾上腺、胰腺的胰岛中有表达，而在肌肉、脂肪组织中无表达，说明ＣＧＡ基因的启动子具 有神经内分泌组织特异性。更多还原

【Abstract】 The vector pGAS1C-lacZ was constructed which contains cga promoter-lacZ and cga promoter-cga antisense cDNA two major parts. The pGAS1C-lacZ was integrated into genome of PC-12 cells and lacZ gene expressed in PC-12 cells. We cut the pGAS1C-lacZ with Zba I and Sal I to get rid of the framework of plasmid pGAS1C-lacZ, then microinjected the other pat which contains antisense DNA into female pronucleus of the fertile mouse eggs. All injected fertile eggs were transplanted into the oviduct of foster so that the injected eggs developed normally. We selected transgenic mice by PCR, and got 14 transgenic founders. The founders were hybridized with normal mice respectively to reproduce the offspring F1 of founders. We executed all the founders and took their adrenal glands to dye with X-Galrespectively. Paraffin sections of the dyed adrenal glands were used to determine the level of lacZ expression in transgenic mice, so we selected two founders which adrenal glands were dyed deeply, and remained their offsprings. Taking different different from the transgenic mice to dye with X-Gal respectively, and we found the lacZ gene expressed in the adrenal gland and pancreas, but not in the muscle and adipose tissues. This result shows the neuroendocrine tissue specific promoter of Chromogranin A gene.更多还原