RRC ID 30908
Author Akatsuka Y, Goldberg TA, Kondo E, Martin EG, Obata Y, Morishima Y, Takahashi T, Hansen JA.
Title Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines.
Journal Tissue Antigens
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.
Volume 59(6)
Pages 502-11
Published 2002-6-1
DOI 10.1034/j.1399-0039.2002.590607.x
PII tan590607
PMID 12445320
MeSH Antibodies, Monoclonal Antigen Presentation Antigens, Surface / genetics B-Lymphocytes / immunology* B-Lymphocytes / metabolism Base Sequence Cell Line Cloning, Molecular / methods* Consensus Sequence DNA Primers DNA, Complementary Flow Cytometry Gene Expression Genetic Vectors Histocompatibility Antigens Class I / genetics* Histocompatibility Antigens Class I / immunology Humans Interferon-gamma / metabolism Molecular Sequence Data Polymerase Chain Reaction Retroviridae / genetics Transfection
IF 1.348
Times Cited 35
DNA material Human HLA-A*02:01:01 cDNA (RDB02866) Human HLA-A*02:06 cDNA (RDB02867) Human HLA-A*02:07 cDNA (RDB02868) Human HLA-A*11:01:01 cDNA (RDB02869) Human HLA-A*26:01 cDNA (RDB02870) Human HLA-A*24:02:01 cDNA (RDB02871) Human HLA-A*31:01:02 cDNA (RDB02872) Human HLA-A*33:03 cDNA (RDB02873) Human HLA-B*07:02:01 cDNA (RDB02874) Human HLA-B*15:01:01 cDNA (RDB02875) Human HLA-B*35:01:01 cDNA (RDB02876) Human HLA-B*40:01:02 cDNA (RDB02877) Human HLA-B*40:02 cDNA (RDB02878) Human HLA-B*44:03:01 cDNA (RDB02879) Human HLA-B*46:01 cDNA (RDB02880) Human HLA-B*51:01:01 cDNA (RDB02881) Human HLA-B*52:01:01 cDNA (RDB02882) Human HLA-C*01:02 cDNA (RDB02883) Human HLA-C*03:03:01 cDNA (RDB02884) Human HLA-C*03:04:01 cDNA (RDB02885) Human HLA-C*04:01:01 cDNA (RDB02886) Human HLA-C*07:02 cDNA (RDB02887) Human HLA-C*08:01 cDNA (RDB02888) Human HLA-C*12:02:02 cDNA (RDB02889) Human HLA-C*14:03 cDNA (RDB02890) Human HLA-A*24:20 cDNA (RDB03365) Human HLA-A*26:03 cDNA (RDB03366) Human HLA-A*03:01 cDNA (RDB03367) Human HLA-A*01:01 cDNA (RDB03368) Human HLA-B*08:01 cDNA (RDB03369) Human HLA-B*38:01 cDNA (RDB03370) Human HLA-B*39:01:01 cDNA (RDB03371) Human HLA-B*48:01 cDNA (RDB03372) Human HLA-B*54:01 cDNA (RDB03373) Human HLA-B*55:02 cDNA (RDB03374) Human HLA-B*59:01 cDNA (RDB03375) Human HLA-C*01:03 cDNA (RDB03376) Human HLA-C*06:02 cDNA (RDB03377) Human HLA-C*14:02:01 cDNA (RDB03378) Human HLA-C*15:02:01 cDNA (RDB03379)