水牛胎儿成纤维细胞转染外源基因的初步研究

Primary investigation on transfection foreign genes of buffalo fetal fibroblasts

研究了水牛胎儿成纤维细胞(buffalo fetal fibroblasts,BFF)转染外源基因后的细胞生物学特性。纯化含有绿色荧光蛋白(green fluorescent protein,GFP)和新霉素抗性基因的线性化载体,用脂质体法转染体外培养3代的BFF细胞,应用抗生素G418筛选2周后挑取转基因抗性细胞集落扩大培养。结果发现:经过G418筛选后细胞增殖活力下降,胰蛋白酶消化法共分离获得162个转基因水牛阳性细胞集落,转移到24孔板扩大培养后,132个(85.2%)细胞集落可以存活,转移到4孔板中后仅有52个(32.1%)细胞集落能继续生长,最终能在35 mm培养皿中大量培养的只有15个(<9.3%)。抗性细胞由梭形、成簇生长变为多角形、不规则形弥漫生长,立体感减弱,细胞生长速度减慢,活力下降。染色体核型异常率增加,10个抗性细胞集落核型正常率平均为61%,显著低于对照组86.7%(P<0.01)。研究结果为建立和优化BFF细胞转染外源基因平台奠定了工作基础。

The growing status of transgenic buffalo fetal fibroblasts （BFF） cells was investigated in this study. The 3^rd buffalo fetal fibroblasts were transfected with lined plasmid contained green fluorescent protein gene and neo gene by Lipofectin reagent. After two weeks of selection culturing in G418, the transgenic cell colonies were collected and detected cytobiology characterization. The results showed that： the proliferation vitality of anti-G418 cell colony was decreased. One hundred and sixty two anti-G418 cell colonies were collected, when transfered to the 24 well plates 132 （85.2%） colonies could survive, and transfered into 4 wells plate just 52 （32.1%） left, and when enlarge cultured in 35 mm plate, just 15 （〈9.3%） colonies left. The anti-G418 colonies cells morphology changing could be seen with normal cells as control which grown on coverslips after hematoxylin and eosin staining. The chromosome karyotype of 10 anti-G418 colonies were checked, and found that 61% chromosome karyotype were normal, which was greatly significant lower than that of normal culturing BFF cells （86.7%, P〈0.01）. These results will be used as a good foundation in production of transgenic cloned buffalo.