Bifunctional Protein Conferring Enhanced Green Fluorescence and Puromycin Resistance

Abstract

A new genetic marker was created in which sequences from enhanced green fluorescent protein were fused to those of puromycin N-acetyl transferase. The resulting fusion protein (EGFP-puro) conferred both green fluorescence and resistance to puromycin when expressed in mammalian cells. The utility of EGFP-puro as a selectable/screenable marker was demonstrated by the ease with which a recombinant guinea pig cytomegalovirus containing EGFP-puro was isolated by a combination of puromycin selection and screening for green fluorescence. We conclude that EGFP-puro is a compact and versatile marker that should prove useful for recombinant virus and transgenic cell line construction, particularly in applications in which coding capacity is limited.

Requisite H2k Role in NK Cell-Mediated Resistance in Acute Murine Cytomegalovirus-Infected MA/My Mice

“…Recombinant virus construction was performed essentially as described (25). Briefly, a deletion mutation spanning the m157 open reading frame (ORF) was introduced into the WT K181 MCMV genome using homologous recombination.…”

“…MCMV targeting sequences (right ϭ 214,490 -215,536 and left ϭ 217,872-218,831) were PCR amplified using highfidelity TripleMaster polymerase mix (Eppendorf) and subcloned into pBluescript II skϪ for sequence verification. Right (ClaI/XhoI) and left (EcoRI/SpeI) restriction fragments were subsequently subcloned into pEGFP-Puro (25). NIH3T3 cells were transfected with the resultant targeting plasmid pTC-⌬m157 and later infected with WT K181 MCMV.…”

Human Cytomegalovirus UL84 Insertion Mutant Defective for Viral DNA Synthesis and Growth

“…A UL84 expression plasmid, pZP13, containing full-length UL84 with its native promoter, was constructed as previously described (21). The enhanced green fluorescent protein (EGFP)-puromycin cassette was described previously (1).…”

“…pBS-UL84 was cleaved with MunI in the middle of the UL84 ORF and then treated with the E. coli DNA polymerase Klenow fragment to generate blunt ends. An EGFP-puromycin fusion protein expression cassette (1) was ligated into the blunt-ended MunI site. The resultant plasmid, pBS-IN84/Ep, was then cleaved with KpnI to release an 11.2-kb fragment containing the EGFP-puromycin cassette flanked by about 4.5 kb of UL84 flanking sequence at both sites.…”

Down-regulation of surface major histocompatibility complex class I by guinea pig cytomegalovirus

“…Guinea pig embryo fibroblast (GEF) cells were cultivated in culture medium consisting of Dulbecco's Modified Essential Medium, 10 % foetal bovine serum (FBS), 50 mg streptomycin ml 21 and 50 U penicillin (BioWhittaker) ml 21 as previously described (McVoy et al, 1997). Virus-infected cells were distinguished from uninfected cells by the use of a recombinant virus, GPCMV/EGFP, which expresses enhanced green fluorescent protein (EGFP) fused to puromycin N-acetyltransferase (Abbate et al, 2001). Cells were mock-infected or infected with GPCMV/EGFP at different m.o.i.…”