EFFICIENT TRANSFECTION OF CHICKEN-CELLS BY LIPOFECTION, AND INTRODUCTION OF TRANSFECTED BLASTODERMAL CELLS INTO THE EMBRYO
EFFICIENT TRANSFECTION OF CHICKEN-CELLS BY LIPOFECTION, AND INTRODUCTION OF TRANSFECTED BLASTODERMAL CELLS INTO THE EMBRYO
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DOI：
10.1002/mrd.1080300404
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发表时间：
1991-12-01
影响因子：
2.5
通讯作者：
GIBBINS, AMV
中科院分区：
文献类型：
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作者：
BRAZOLOT, CL;PETITTE, JN;GIBBINS, AMV
鸡胚盘细胞（CBCs）和原代鸡成纤维细胞（PCF）已经用编码大肠杆菌β-半乳糖苷酶（β-gal）的多种lacZ构建体脂质体转染。 使用含有小鼠热休克蛋白68基因（hsp 68）启动子的报告基因构建体（phspPTlacZpA）来建立有效脂质转染的条件。 通过在6.2 μ g Lipofectin（TM）（来自Bethesda Research Laboratories的阳离子脂质体制剂）和1.55-3.1 μ g DNA/mL DMEM的混合物中孵育细胞3.5小时，有效地转移环状或线性质粒形式或单独作为报道序列的构建体。 这些脂质转染条件用于转移报告基因构建体（pCBcMtlacZ），以及由于劳斯肉瘤病毒加鸡β-肌动蛋白（pmiwZ）或巨细胞病毒（pMaori 3）启动子而显示组成型表达的构建体。pH4.3或7.4时， phspPTlacZpA在鸡细胞中的表达似乎不需要小鼠hsp 68启动子的特异性诱导，而pCBcMtlacZ的表达需要用150 μ M ZnCl 2处理细胞6-12小时。 在对悬浮培养或平板接种的CBC进行脂质转染后观察到细菌β-gal活性。 对于CBCs，脂质转染的效率至少为1/25，通过脂质转染处理开始后16-24小时显示具有β-gal活性的细胞比例来判断;这些事件可以代表构建体的瞬时或稳定掺入。 将脂质转染的CBC注射到阶段X（Eyal-Giladi和Kochav，1976）鸡胚胎的胚下腔中，其方式已经显示对于未处理的CBC产生体细胞和种系嵌合体（Petitte等人，1990年）。 孵育65小时后，在11期胚胎的前脑、头部外胚层和心室中观察到表达β-gal活性的细胞（Hamburger和汉密尔顿，1951）。 在其他情况下，在注射后24、48和65小时，在单个细胞和表达细胞的病灶中胚外检测到细菌β-gal活性。 将Lipofectin（TM）：DNA混合物直接注射到胚胎中后，几乎没有（如果有的话）单个表达细胞，也没有检测到病灶。
Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2-mu-g Lipofectin(TM) (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1-mu-g DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn2+-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters.Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150-mu-M ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct. Plated PCFs were lipofected as well, with stable incorporation of the gene construct indicated in 10% of positive events.Lipofected CBCs were injected into the subgerminal cavity of stage X (Eyal-Giladi and Kochav, 1976) chicken embryos in a manner that has been shown to produce somatic and germline chimeras for untreated CBCs (Petitte et al., 1990). After 65 h of incubation, cells expressing beta-gal activity were observed in the prosencephalon, head ectoderm, and ventricle of the heart of a stage 11 (Hamburger and Hamilton, 1951) embryo. In other cases, bacterial beta-gal activity was detected extraembryonically, both in individual cells and in foci of expressing cells, 24, 48, and 65 h after injection. Few, if any, single expressing cells and no foci were detected following injection of the Lipofectin(TM): DNA mixture directly into the embryo.Refinement of these procedures could contribute to the development of transgenic poultry, without reliance on retroviral vectors for DNA transmission or incorporation.