Transient selection during vaccinia virus recombination with insertion vectors without selectable markers.
Isolation of recombinant viruses after DNA transfection remains the most time-consuming and labor-intensive feature in the production of vaccinia virus (VV) recombinants. This procedure has been aided by the design and incorporation of both positive and negative selectable markers into the insertion vectors. Use of these selectable markers is not always feasible or desirable. In the absence of selectable markers, individual plaque characterization is required. A method of transient selection during transfection and recombination is described that does not require that any selectable marker be present in the insertion vector sequence. This method relies on the co-transfection of a guanine phosphoribosyl transferase (gpt) selectable marker on an unlinked plasmid into VV-infected cells pretreated for gpt selection. Following recombination, the output virus can be plated on cells without any further selection and screened for recombinant virus. While the total recombinant virus population is slightly reduced, the total progeny virus is reduced even further, resulting in more than sevenfold enhancement in the frequency of recombinant virus, which allows for more efficient identification.
Kurilla
M G
MG
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908, USA. mgk2r@virginia.edu
D002130:Calcium Phosphates; D011232:Chemical Precipitation; D004274:DNA, Recombinant; D005819:Genetic Markers; D005822:Genetic Vectors; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009173:Mycophenolic Acid / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics* / Q000302:isolation & purification*
D002130:Calcium Phosphates; D004274:DNA, Recombinant; D005819:Genetic Markers; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D007041:Hypoxanthine Phosphoribosyltransferase; D009173:Mycophenolic Acid; C485829:calcium phosphate, dibasic, anhydrous