Variable Efficiency of Retroviral-Mediated Gene Transfer into Early-Passage Cultures of Fetal Lamb Epithelial,Mesenchymal,and Neuroectodermal Tissues

Abstract

The relative efficiency of retroviral-mediated gene transfer into early-passage cultures of different tissues of fetal lamb was investigated. Monolayer cultures prepared by plating 1 × 10⁶ cells were infected with the Moloney murine leukemia (MoMLV)-based vector pZIP Neo at a multiplicity of infection (moi) of approximately 1 pfu per 2 × 10² recipient cells prior to selection for neomycin resistance. At the low moi used, cells from different tissues showed marked differences in efficiency of colony formation in the descending order: brain > kidney > muscle, lung > skin. Brain cells were transduced at least an order of magnitude more efficiently than other cell types, despite the doubling time of brain cell cultures being five times as long. Cultures were analyzed by morphological and immunocytological criteria to determine whether any particular cell types were transduced. A wide variety of morphologially distinct neuron-like and glial-like brain cells were neomycin resistant. The majority of muscle cell colonies were myogenic. Approximately half of the large kidney colonies were epithelial-like. The majority of lung colonies consisted of fibroblasts. The results suggest that cells originating from the surface embryonic germ layer (ectoderm) and/or occupying positions near the fetal external surface have a markedly lower susceptibility to retroviral-mediated gene transduction.

Overview summary

Moloney murine leukemia-based retroviral vectors have been used to transfer, integrate, and express foreign genes efficiently in a wide variety of mammalian cells in vitro and in vivo. However, there have been few comparative investigations of the relative efficiency of transduction into early passage cultures of different tissues. John has found that the susceptibility of fetal tissues to infection by low-titer retroviral vector showed a marked variability, which may depend upon the embryonic germ layer from which the tissues originate and/or their proximity to the fetal external surface.