A novel method for generating stable high-level transfectants involving direct immunomagnetic selection for cell-surface epitopes: expression of HLA class-II genes in HeLa cells.
A versatile method that allows efficient detection and selection of both transient and stable transfectants expressing exogenous cell-surface molecules is described and used to generate stable HeLa transfectants expressing each of the human HLA class-II isotypes, specifically the DR1, DQw8 and DPw2 heterodimers. The method combines use of the strong mammalian expression vector, CDM8, and a highly efficient transfection protocol with the powerful technique of immunomagnetic selection. It offers significant advantages in comparison to standard procedures involving co-selection with drug-resistance markers. The transfection efficiency can be assessed 60 h after transfection rather than after three weeks of drug selection. Repeated rounds of immunomagnetic selection applied over the subsequent ten days result in homogeneous populations which express the surface marker of interest stably at high levels, making further subcloning or fluorescence-activated cell sorting unnecessary. Any number of surface products can be transfected into the same cell, the only limitation being the availability of specific monoclonal antibodies (a DP/DR double transfectant is described expressing four exogenous gene products simultaneously). The high sensitivity of immunomagnetic selection and its applicability to large samples allows rescue of transfectants present at very low frequencies. Finally, the technique can be used as a coselection procedure, by analogy with drug coselection, to achieve expression even of non-cell surface products.
Kappes
D J
DJ
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Strominger
J L
JL
Monos
D S
DS
D003001:Cloning, Molecular; D005434:Flow Cytometry; D015870:Gene Expression / Q000235:genetics; D005802:Genes, MHC Class II; D005822:Genetic Vectors / Q000235:genetics; D006682:HLA-DP Antigens / Q000032:analysis* / Q000235:genetics; D006683:HLA-DQ Antigens / Q000032:analysis* / Q000235:genetics; D015798:HLA-DR1 Antigen / Q000032:analysis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D012397:Rosette Formation; D014162:Transfection / Q000235:genetics
D006682:HLA-DP Antigens; D006683:HLA-DQ Antigens; C109756:HLA-DQ8 antigen; D015798:HLA-DR1 Antigen