摘要
目的建立四环素诱导调控的MT转基因小鼠模型,为血管瘤试验研究及MT致瘤机理研究奠定基础。方法通过PCR方法从鸡的基因组序列中克隆绝缘子元件;构建四环素诱导调控的条件化转基因质粒,并将其调控元件和受控元件串联成一个载体,在两元件之间插入绝缘子,以减轻两者之间的相互干扰。为提高转基因的表达效率,在转基因盒的上游亦插入绝缘子元件;将MT基因亚克隆至此载体;转基因载体功能行细胞瞬转试验及半定量逆转录PCR验证后,将其在体外扩增、酶切、回收,行小鼠受精卵原核注射,获得转染MT基因阳性小鼠;强力霉素体外诱导部分阳性鼠MT基因表达,使其具有血管瘤表型。结果绝缘子元件成功克隆;成功构建条件化转基因载体;体外试验证实目的基因的表达受强力霉素的严格调控;通过原核注射,获得5只转基因阳性鼠;2只行体外诱导2月后,1只出现血管瘤表型,逆转录PCR检测证实MT表达。其余3只阳性小鼠在传代建系中。结论条件化MT转基因小鼠模型成功构建,此小鼠模型能够为血管瘤的试验研究及MT致瘤机理的体内研究提供一定的基础。
Objeictive To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo. Methods Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two dements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene caasette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts （target） in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR. Results The conditional transgenic vector was constructed succesefully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjeetion. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding. Conclusion Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2006年第3期260-264,共5页
Chinese Journal of Medical Genetics
基金
国家自然科学基金(30371546)
上海市重点学科建设资助项目(Y0203)~~
