Cloning of an unidentified T-cell receptor alpha chain gene from a vesicular stomatitis virus-specific helper T-cell clone.
A vesicular stomatitis virus glycoprotein-specific, class II restricted, CD4+ T-cell clone was obtained and the unidentified T-cell receptor alpha chain cloned in order to establish a T-cell receptor (TCR) alpha chain transgenic mouse line. Polymerase chain reaction (PCR) strategies have been developed to clone TCR chain genes starting from T-cell cDNA. There remain difficulties, however, in cloning the functional TCR alpha chain due to the complexity of the protocols applied if the variable (V) alpha region rearranged is not known. The strategy described here allows the identification and cloning of alpha chains that are not recognized by any of the anti-V alpha monoclonal antibodies available: three 5' consensus V alpha primers designed from all known V alpha gene sequences and a primer specific for the constant (C) alpha region were used and the PCR product sequenced. The T-cell clone displayed two alpha gene rearrangements, only one of which giving rise to a functional protein. The alpha chain used by the T-cell clone contained a V alpha 3.1 and a J alpha region which has been described only at the genomic level, but never in a functional TCR. The complete alpha chain gene was cloned by enriching the alpha chain-encoding cDNA by ligation-anchored PCR and using an alpha specific primer pair. The use of the present method, even if the sequence of the 5' untranslated (UT) region of the alpha chain is not known, is also discussed.
Ghelardi
E
E
Institute for Experimental Immunology, Department of Pathology, University of Zürich, Switzerland.
Burkhart
C
C
Senesi
S
S
Hengartner
H
H
Freer
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D005796:Genes; D008562:Membrane Glycoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000166:cytology / Q000276:immunology*; D014721:Vesicular stomatitis Indiana virus / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000276:immunology*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D014759:Viral Envelope Proteins; D004247:DNA