Expression cloning of neurotrophic factors using Xenopus oocytes.
We have explored the potential for cloning novel neurotrophic factor cDNAs via assay of neurotrophic activities following expression in Xenopus oocytes. In this report, we describe the successful application of the method to tract rat ciliary neurotrophic factor (CNTF) activity from mRNA purified from cultured cells and from mRNA synthesized by in vitro transcription of a cDNA library. Rat C6 glioma cells, which had been previously shown to have CNTF-like activity (Westermann et al., 1988), were used as source material. We tested protein extracts of C6 cells using an in vitro assay of primary neurons from the chick ciliary ganglion (CCG assay) and detected a CNTF-like activity. RNA isolated from C6 cells was shown to direct the synthesis of the activity following microinjection into Xenopus oocytes and one-step fractionation of Xenopus extract. C6 mRNA was size-fractionated, and fractions encoding CNTF-like activity were cloned into a lambda phage vector at a site distal to a T7 promoter. Synthetic RNA transcribed from total library DNA was injected into Xenopus oocytes, and a CNTF-like activity in the oocyte extract was detected by the CCG assay. Further fractionation of library clones narrowed the presence of the clone encoding the CNTF-like activity to a pool containing 20,000 members. The presence of a full-length CNTF cDNA clone in this pool and partial clones in other pools was confirmed by Polymerase Chain Reaction (PCR) using oligonucleotides from the rabbit CNTF cDNA (Lin et al., 1989) as primers.(ABSTRACT TRUNCATED AT 250 WORDS)
Lam
A
A
California Biotechnology, Inc., Mountain View 94043.
Kloss
J
J
Fuller
F
F
Cordell
B
B
Ponte
P A
PA
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D015723:Gene Library; D007267:Injections; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009865:Oocytes / Q000502:physiology*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000502:physiology; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis / Q000235:genetics*
D020934:Ciliary Neurotrophic Factor; D015335:Molecular Probes; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger