Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron.
The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.
González
José M
JM
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Pénzes
Zoltan
Z
Almazán
Fernando
F
Calvo
Enrique
E
Enjuanes
Luis
L
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D016679:Genome, Viral; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism; D014162:Transfection; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000472:pathogenicity*
D018076:DNA, Complementary; D012367:RNA, Viral