Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.
The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA.
Duan
L
L
Dorrance H. Hamilton Laboratories, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
Pomerantz
R J
RJ
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D015483:HIV Antibodies / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics* / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011131:Polyribonucleotides / Q000138:chemical synthesis; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D014764:Viral Proteins / Q000276:immunology
D015483:HIV Antibodies; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011131:Polyribonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014764:Viral Proteins