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.

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. -->
Substance Nomenclature: 0 (DNA Primers)
0 (DNA, Recombinant)
0 (DNA, Viral)
Entry Date(s): Date Created: 19960210 Date Completed: 19961206 Latest Revision: 20060421
Update Code: 20221213
DOI: 10.1089/hum.1996.7.3-343
PMID: 8835221
Autor: Gerard CJ; Department of Gene Therapy, SyStemix, Inc., Palo Alto, CA 94304, USA., Arboleda MJ, Solar G, Mulé JJ, Kerr WG
Jazyk: angličtina
Zdroj: Human gene therapy [Hum Gene Ther] 1996 Feb 10; Vol. 7 (3), pp. 343-54.
DOI: 10.1089/hum.1996.7.3-343
Abstrakt: 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.
Databáze: MEDLINE
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