A study in development of transgenic cattles for the use of hybrid bioartifical liver.

在异种肝脏体外生物人工肝灌注系统的研究中，转基因技术的主要目的是敲除异种蛋白的基因，将人蛋白的基因转导到动物体内。我们初步计划将人白蛋白cDNA转导到猪生殖细胞中，制备能分泌人白蛋白的猪肝，并将其用于生物人工肝支持。我们获得了人白蛋白cDNA （hAlb）和EGFP（增殖型绿色荧光蛋白）基因pCX-hAlb-EGFP。我们先用脂肪转染法转染NIH3T3细胞，然后用经典的显微注射法转染小鼠生殖细胞，再用胞浆内单精子注射（ICSI）法转染猪生殖细胞，目的是诱导人白蛋白在培养细胞和动物肝脏中的表达。结果表明，NIH3T3细胞在Northern杂交分析中表现为紫外线亮白并表达人白蛋白，并获得了转基因猪，其皮肤在PCR分析中表现为紫外线亮白并整合人白蛋白基因。虽然猪出生时死亡，但这是ICSI法在TG猪中表达EGFP和人白蛋白的首次划时代报道。

In research on extracorporeal bioartificial liver perfusion system using xenogeneic liver, the chief purpose of the transgenic technique is to knock out genes of xenogeneic proteins, and transduce genes of human proteins into the animal. As an initial approach, we planned to transduce human albumin cDNA into porcine germ cells to produce porcine liver which can secret human albumin for the purpose of utilizing it for bioartificial liver support, we produced a gene of human albumin cDNA (hAlb) and EGFP (enhanced green fluorescent protein) gene, pCX-hAlb-EGFP. We transfected it initially into NIH3T3 cells using lipofection method, secondly into mouse germ cells by classic microinjection method, and thirdly into porcine germ cells by intracytoplasmic sperm injection (ICSI) method in order to induce expression of human albumin in the cultured cells and animal liver. As the results, NIH3T3 cells showed ultraviolet lightening and expressed human albumin in Northern hybridization analysis, and a transgenic pig was produced of which the skin showed ultraviolet lightening and integration of human albumin gene in PCR analysis. It was a pity that the pig was born dead, but this was initial epoch-making report of expression of EGFP and human albumin in TG pig produced by ICSI method.