ISSN:
1040-452X
Keywords:
Transfection
;
Chimeras
;
β-galactosidase
;
lacZ
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli β-galactosidase (β-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 μg LipofectinTM (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 μg 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 β-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters.Endogenous chicken β-gal and transferred bacterial β-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 μM ZnCl2. Bacterial β-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 b̃-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 β-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 β-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 LipofectinTM: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.
Additional Material:
3 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/mrd.1080300404
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