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

摘要

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.