A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96-well format PCR and fluorescent detection system universal for MMLV-based proviruses.
The polymerase chain reaction (PCR) is an extremely sensitive assay that has many uses in retroviral-mediated gene transfer protocols. Because the majority of retroviral vectors used in current gene transfer protocols are based on the Moloney-murine leukemia virus (MMLV), we have designed primers which amplify a region of the psi packaging sequence from all MMLV retroviruses tested. This assay detects gene transfer by all MMLV-based vectors and is especially useful for the laboratory that routinely screens a number of different retroviruses for their gene transfer efficiency. Furthermore, we present here a novel technique for harvesting single colonies derived from hematopoietic stem/progenitor cells growing in methylcellulose medium that expedites and substantially improves the resulting quantitative estimates of retroviral transduction frequencies. This technique utilizes a conventional 96-well format and, when coupled with a fluorescence-based post-PCR detection system, makes it unnecessary to run agarose gels to visualize the PCR product. This system of PCR product detection, which uses the 5'-->3' exonuclease activity of Taq DNA polymerase to cleave a fluorescently labeled probe during each round of PCR amplification, is fast, convenient, and at least as sensitive as an ethidium bromide-based detection system when used in conjunction with our universal PCR assay.
Gerard
C J
CJ
Department of Gene Therapy, SyStemix, Inc., Palo Alto, CA 94304, USA.
Arboleda
M J
MJ
Solar
G
G
Mulé
J J
JJ
Kerr
W G
WG
D000208:Acute Disease; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000295:instrumentation; D002999:Clone Cells / Q000821:virology; D017931:DNA Primers; D004274:DNA, Recombinant / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D005470:Fluorometry; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D006412:Hematopoietic Stem Cells / Q000821:virology*; D006801:Humans; D007951:Leukemia, Myeloid / Q000473:pathology; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004274:DNA, Recombinant; D004279:DNA, Viral