大分子BAC基因打靶载体转染鸡胚原始生殖细胞

Macromolecule BAC targeting vector for transfection of chicken embryo primordial germ cells

背景：利用原始生殖细胞技术可以方便地将各种外源基因转入鸡体内,以较快的速度获得转基因鸡,但到目前为止原始生殖细胞的转染效率仍然很低,且很难进行较大基因片段的转染。目的：拟实现大分子BAC载体对鸡胚原始生殖细胞的转染。设计、时间及地点：基因水平的细胞学体外观察,于2006-08/2008-07在广东省佛山大学完成。材料：种蛋来自华南农业大学鸡场粤黄鸡群,受精蛋在37.5℃、相对湿度为65%的条件下孵化68~72h。BAC-TDN-GID载体由佛山大学分子生物学实验室保存。方法：以rRNA基因及其间隔序列作为同源重组引导序列,构建含GFP基因和hIFN基因的大分子BAC基因打靶载体,将鉴定为阳性的载体进行扩增培养并大量抽提。取课题组孵化至第19期的粤黄鸡胚,挑取其生殖嵴或性腺,胰酶消化法获得原始生殖细胞,并辅以饲养层和生长因子进行培养。采用脂质体介导法在原始生殖细胞内进行基因转移,对存活的细胞进行目的基因DNA水平和RNA水平鉴定。结果：第19期鸡胚性腺原始生殖细胞体外培养48h呈桑椹状或者椭圆状,72h形成细胞集落,过碘酸染色呈紫红色。分子量达30kb的大分子转基因载体BAC-TDN-GID转染鸡胚原始生殖细胞48h可见荧光细胞,表明转染成功。经PCR和RT-PCR鉴定,证实扩增的产物大小与预期结果大致相吻合,目的基因hIFN已经整合进入原始生殖细胞基因组,并已开始表达。结论：实验成功将大分子BAC载体上的外源基因转染入体外分离培养的鸡胚原始生殖细胞。

BACKGROUND： Using the primordial germ cells （PGCs） technology, the foreign genes can be easily transferred into the chicken to get the transgenic chicken in shorter time. However, till now, the transfection efficiency of PGCs is very low and it is specially difficult that longer gene fragment is transfected into PGCs. OBJECTIVE： To achieve the transfection of exogenous gene of macromolecule BAC vector into PGCs. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Foshan University from August 2006 to July 2008. MATERIALS： Hatching egg was obtained from Yuehuang chicken at the chicken farm of Southern China Agriculture University. Fertile egg was hatched at 37.5℃ under relative humidity of 65% for 68-72 hours. BAC-TDN-GID vector was maintained at the Laboratory of Molecular Biology, Foshan University. METHODS： Using the DNA sequences of rRNA repeating gene family and its internal transcribed spacers as homologous recombination directed sequences （HRDS）, the vector of macromotecule BAC containing GFP gene and hlFN gene had been constructed. The constructed vector containing GFP gene and hlFN gene was identified and then the positive clone would be expanded cultured and extracted on a large scale. The genital ridge or the gonad of stage 19 embryos were picked and got. PGCs were got by digestion with trypsin. PGCs were incubated on feeder layer cells with growth factors. The vector was transferred into PGCs by using the liposome mediate method. The survival cells were identified in the levels of DNA and RNA. RESULTS： The PGCs from the genital ridge or the gonad of stage 19 embryos were mulberry-like shape or oval at 48 hours. Cell colony was formed and prunosus was found following periodic acid-Schiff staining at 72 hours. The BAC-TDN-GID of macromolecule vector whose molecular weight is 30 kb was successfully transferred into PGCs by the existence of fluorescence cells. Polymerase chain reaction and reverse transcription-polymerase chain reaction had confirmed that the fragment of amplified products was identical to expecting results. Target gene hlFN had been inserted into the genome of PGCs and expressed. CONCLUSION： The exogenous gene on the macromolecule BAC vector has been transferred into in vitro cultured PGCs.