Production and design of more effective avian replication-incompetent retroviral vectors.
Retroviral vectors have been invaluable tools for studies of development in vertebrates. Their use has been somewhat constrained, however, by the low viral titers typically obtained with replication-incompetent vectors, particularly of the avian type. We have addressed this problem in several ways. We optimized the transient production of avian replication-incompetent viruses in a series of cell lines. One of the optimal cell lines was the mammalian line 293T, which was surprising in light of previous reports that avian viral replication was not supported by mammalian cells. We also greatly increased the efficiency of viral infection. Pseudotyping with the vesicular stomatitus virus G (VSV-G) protein led to an over 350-fold increase in the efficiency of infection in ovo relative to infection with virus particles bearing an avian retroviral envelope protein. To further increase the utility of the system, we developed new Rous sarcoma virus (RSV)-based replication-incompetent vectors, designed to express a histochemical marker gene, human placental alkaline phosphatase, as well as an additional gene. These modified retroviral vectors and the VSV-G pseudotyping technique constitute significant improvements that allow for expanded use of avian replication-incompetent viral vectors in ovo.
Chen
C M
CM
Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA.
Smith
D M
DM
Peters
M A
MA
Samson
M E
ME
Zitz
J
J
Tabin
C J
CJ
Cepko
C L
CL
D000818:Animals; D001354:Avian Leukosis Virus / Q000235:genetics; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000821:virology; D002460:Cell Line; D002642:Chick Embryo; D003673:Defective Viruses / Q000235:genetics*; D005347:Fibroblasts / Q000821:virology; D005822:Genetic Vectors / Q000096:biosynthesis; D006801:Humans; D008562:Membrane Glycoproteins; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids; D011506:Proteins / Q000378:metabolism; D012160:Retina / Q000821:virology; D012190:Retroviridae / Q000235:genetics*; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D014759:Viral Envelope Proteins / Q000235:genetics; D014779:Virus Replication; D014921:Wings, Animal / Q000821:virology
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011506:Proteins; D014759:Viral Envelope Proteins; C055324:phospholipase A2-activating protein