Production of pseudotyped retrovirus and the generation of proviral transgenic zebrafish.
This chapter describes a method for generation of the high-titer pseudotyped Moloney murine leukemia virus (MLV) that efficiently infects zebrafish embryos (i.e., more than 25 retroviral copies per cell). Injection techniques are also described for production of the retrovirus-infected mosaic "founder" fish. We describe a quantitative PCR (qPCR)-based assay as a quick way to assess the infectivity after each round of viral production and injection. Most of the required equipment is commercially available and commonly present in most research laboratories.
Jao
Li-En
LE
Cell and Developmental Biology, Vanderbilt University, TN, Nashville, USA.
Burgess
Shawn M
SM
D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D046528:Chimerism; D004247:DNA / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D016678:Genome / Q000235:genetics; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000821:virology*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D004247:DNA