Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines

Y. Akatsuka, T. A. Goldberg, E. Kondo, E. G. Martin, Y. Obata, Y. Morishima, T. Takahashi, J. A. Hansen

Research output: Contribution to journalArticle

35 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)502-511
Number of pages10
JournalTissue Antigens
Volume59
Issue number6
DOIs
Publication statusPublished - Jun 2002
Externally publishedYes

Fingerprint

Gibbon ape leukemia virus
Organism Cloning
Complementary DNA
Retroviridae
Cell Line
Polymerase Chain Reaction
HLA-C Antigens
MHC Class I Genes
Puromycin
HLA-A Antigens
HLA-B Antigens
5' Untranslated Regions
Linkage Disequilibrium
3' Untranslated Regions
Clone Cells
Alleles
Lymphocytes
T-Lymphocytes
Genes

Keywords

  • Cloning
  • HLA class I
  • PCR
  • Retrovirus vector

ASJC Scopus subject areas

  • Immunology
  • Cell Biology

Cite this

Akatsuka, Y., Goldberg, T. A., Kondo, E., Martin, E. G., Obata, Y., Morishima, Y., ... Hansen, J. A. (2002). Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines. Tissue Antigens, 59(6), 502-511. https://doi.org/10.1034/j.1399-0039.2002.590607.x

Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines. / Akatsuka, Y.; Goldberg, T. A.; Kondo, E.; Martin, E. G.; Obata, Y.; Morishima, Y.; Takahashi, T.; Hansen, J. A.

In: Tissue Antigens, Vol. 59, No. 6, 06.2002, p. 502-511.

Research output: Contribution to journalArticle

Akatsuka, Y, Goldberg, TA, Kondo, E, Martin, EG, Obata, Y, Morishima, Y, Takahashi, T & Hansen, JA 2002, 'Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines', Tissue Antigens, vol. 59, no. 6, pp. 502-511. https://doi.org/10.1034/j.1399-0039.2002.590607.x
Akatsuka, Y. ; Goldberg, T. A. ; Kondo, E. ; Martin, E. G. ; Obata, Y. ; Morishima, Y. ; Takahashi, T. ; Hansen, J. A. / Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines. In: Tissue Antigens. 2002 ; Vol. 59, No. 6. pp. 502-511.
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