遗传学报
遺傳學報
유전학보
ACTA GENETICA SINICA
2001年
5期
398-404
,共7页
嗜铬颗粒蛋白A基因%反义RNA
嗜鉻顆粒蛋白A基因%反義RNA
기락과립단백A기인%반의RNA
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。
構建小鼠嗜鉻顆粒蛋白A（Chromogranin A，CGA）基因的反義DNA載體pGAS1C-lacZ。用電穿孔的方法將該載體轉化大鼠腎上腺髓質細胞瘤細胞繫PC-12，X-Gal染色後證明位于CGA基因啟動子下遊的報告基因lacZ已經錶達。用限製性內切酶除去載體的質粒骨架後，顯微註射入供體昆明小鼠的受精卵中，隨後將註射過DNA的受精卵移植入假母的輸卵管中，完成正常的胚胎髮育。用PCR的方法篩選假母產下的小鼠，得到CGA基因反義RNA轉基因首建鼠14隻。將首建鼠分彆與正常昆明鼠交配，產生後代。取首建鼠的腎上腺進行X-Gal染色，組織用于石蠟切片，根據各鼠腎上腺切片的藍色深淺判定轉入基因錶達量的高低，篩選到兩隻錶達量高的首建鼠，留下它們的後代。取轉基因鼠的各種組織用于X-Gal染色，髮現報告基因在腎上腺、胰腺的胰島中有錶達，而在肌肉、脂肪組織中無錶達，說明CGA基因的啟動子具有神經內分泌組織特異性。
구건소서기락과립단백A（Chromogranin A，CGA）기인적반의DNA재체pGAS1C-lacZ。용전천공적방법장해재체전화대서신상선수질세포류세포계PC-12，X-Gal염색후증명위우CGA기인계동자하유적보고기인lacZ이경표체。용한제성내절매제거재체적질립골가후，현미주사입공체곤명소서적수정란중，수후장주사과DNA적수정란이식입가모적수란관중，완성정상적배태발육。용PCR적방법사선가모산하적소서，득도CGA기인반의RNA전기인수건서14지。장수건서분별여정상곤명서교배，산생후대。취수건서적신상선진행X-Gal염색，조직용우석사절편，근거각서신상선절편적람색심천판정전입기인표체량적고저，사선도량지표체량고적수건서，류하타문적후대。취전기인서적각충조직용우X-Gal염색，발현보고기인재신상선、이선적이도중유표체，이재기육、지방조직중무표체，설명CGA기인적계동자구유신경내분비조직특이성。
The vector pGASlC-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 pGASlC-lacZ with Xba Ⅰ and Sal Ⅰ to get rid of the framework of plasmid pGAS1C-lacZ, then microinjected the other part 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-Gal respectively. 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 tissues 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.
