Retrovirol gene transfer in Xenopus cell lines and embryos.
A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three different Xenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10(8) cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the beta-galactosidase gene into the neural tube lumen of 24-h embryos yielded beta-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer in Xenopus embryos and cell lines.
Department of Pediatrics and Center for Molecular Genetics, La Jolla, California 92093, USA.
J C
JC
Burns
L
L
McNeill
C
C
Shimizu
T
T
Matsubara
J K
JK
Yee
T
T
Friedmann
B
B
Kurdi-Haidar
E
E
Maliwat
C E
CE
Holt
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002461:Cell Line, Transformed; D017931:DNA Primers; D004285:Dogs; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D016662:Virus Integration; D014981:Xenopus / Q000196:embryology
D017931:DNA Primers