RETROVIRAL GENE-TRANSFER IN XENOPUS CELL-LINES AND EMBRYOS

A new class of retroviral vector pseudotypes have an expanded host spe cies range and can be concentrated to high titers by ultracentrifugati on. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis v irus substituted for the amphotropic envelope protein. We tested (a) t he ability of pseudotyped (pantropic) and unmodified (amphotropic) vec tors to stably infect three different Xenopus laevis cell lines, inclu ding one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate forei gn gene expression in the embryonic CNS. Expression of the neomycin ph osphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amph otropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stag e 10, 20, and 25 embryos microinjected in the blastocoel or neural tub e cavities with concentrated pantropic vector (10(8) cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector contai ning the coding sequence for the beta-galactosidase gene into the neur al tube lumen of 24-h embryos yielded beta-galactosidase expressing ce lls in the brain. Thus, retroviral vectors provide an additional appro ach to existing strategies for gene transfer in Xenopus embryos and ce ll lines.