title: Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines.
abstract: Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Akatsuka
authors.forename: T A
authors.initials: TA
authors.lastname: Goldberg
authors.forename: E
authors.initials: E
authors.lastname: Kondo
authors.forename: E G
authors.initials: EG
authors.lastname: Martin
authors.forename: Y
authors.initials: Y
authors.lastname: Obata
authors.forename: Y
authors.initials: Y
authors.lastname: Morishima
authors.forename: T
authors.initials: T
authors.lastname: Takahashi
authors.forename: J A
authors.initials: JA
authors.lastname: Hansen
mesh_terms: D000911:Antibodies, Monoclonal; D017951:Antigen Presentation; D000954:Antigens, Surface / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D017931:DNA Primers; D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; D007371:Interferon-gamma