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

Abstract

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 ψ 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.

Overview summary

The polymerase chain reaction (PCR) analysis of colonies of clonogenic cells growing in methylcellulose medium is a routine procedure to estimate the frequency of retroviral transduction into hematopoietic stem/progenitor cells. This study describes a sensitive assay system that takes advantage of the standard 96-well format to expedite the processing of single methylcellulose colonies. Assay sensitivity is dependent on a PCR primer pair which amplifies a region of the ψ packaging sequence of all Moloney-based retroviruses tested. Using this primer pair, we present the optimized PCR conditions for the analysis of single colonies of clonogenic cells growing in methylcellulose medium as well as the conditions for a semiquantitative bulk PCR assay to estimate the transduction frequency immediately following the transduction protocol. This PCR primer pair, along with the capability for more rapid screening of hematopoietic stem/progenitor colonies, is especially useful for the laboratory that is screening a number of different retroviral constructions for their transduction efficiency.