Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo

Abstract

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 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Transgenesis in chickens

“…Neither the method of lipofection (Brazelot et al, 1991) nor of calcium phosphate precipitation (Kopchick et al, 1991) gave effective levels of transfection. In the latter, more extensive experiments, 0.2% (3/1500) of chickens reared from injected embryos were found to possess foreign DNA sequences (specifying bGH) but the animals did not transmit the gene to their progeny.…”

“…The second component of this technique, transfection of donor blastodermal cells, has been investigated by Brazelot et al, (1991). Efficient transfection of blastodermal cells (1:25) was obtained by lipofection of various lacZ gene constructs.…”

In ovo transfection of chicken embryos using cationic liposomes

“…Brazolot et al (1991) noted loci of cultured chicken blastodermal cells expressing a reporter gene 10 days post-transfection in the absence of selection. To investigate the efficiency of stable integration or the maintenance of plasmid DNA as an episome in vitro, CEF were transfected with pRSVL and luciferase activity was assayed during passage of the CEF culture for 24 days without drug selection (Fig.…”

Open reading frame L1 of Marek's disease herpesvirus is not essential for in vitro and in vivo virus replication and establishment of latency.

“…Cells were fixed for 5 min in 0n05 M phosphate buffer pH 7n4 containing 0n2% (v\v) glutaraldehyde, 2 % (v\v) formaldehyde and 2 mM MgCl # followed by three 5 min washes with a solution of 2 mM MgCl # and 0n02 % NP40 in 0n05 M phosphate buffer pH 7n4. The cells were incubated overnight at 37 mC in a solution containing 0n5 mg\ml X-Gal (stock solution 20 mg\ml X-Gal DMSO), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl # in 0n05 M phosphate buffer pH 7n4 (Brazolet et al, 1991).…”