Fig. 5. Indirect immunofluorescence and FACS analysis of HLA expression of a HAT resistant population derived from a cotransfection of Ltk"~ cells with human genomic gene JY158 and HSV-tk. The panel presents specific staining of the transfected cell population with an anti-HLA-A, B, C antibody (W6/32) compared to control.
amount of DNA incorporated into the majority of primary transfectants necessitates the use of this material as donor DNA for a second round of gene transfer and cell sorting. This is performed identically to the first round. The secondary transfectants may contain from between 30 and 100 kbp of donor DNA, and serve as starting material for the cloning of the surface antigen gene in phage or cosmid vectors. These procedures have been detailed in several chapters in this volume and in another volume of this series.18
A number of surface antigen genes have been cloned by this strategy and include the human transferrin receptor,8 nerve growth factor receptor,25 T cell antigen T8,7 and the human myeloid antigen gpl50.26 Other workers have cloned the T4 and T8 T cell antigens using this transfection approach, but performing initial selections with an antibody based resetting assay rather than the FACS.9'27 It is noteworthy that of the genes
25 M. V. Chao, M. A. Brothwell, A. H. Ross, H. Koprowski, A. A. Lanahan, C. R. Buck, and A. Sehgal, Science 232, 518 (1986).
26 A. T. Look, S. C. Peiper, M. B. Rebeutisch, R. A. Ashman, M. T. Rousell, C. W. Rettenmier, and C. J. Sherr, J. Clin. Invest. 75, 569 (1985).
27 P. J. Maddon, D. R. Littman, M. Godfrey, D. E. Maddon, L. Chess, and R. Axel, Cell 42, 93(1985).
cloned by this method, only the transferrin receptor is normally expressed on fibroblasts. Using the transfection/selection procedures outlined here it should be possible to clone a variety of receptor genes by using fluorescein-ated ligands as well as species-specific antibodies.
Mouse cells containing human surface antigen genes introduced by cell hybridization or DNA transfection can be labeled by indirect immunofluorescence and isolated using the FACS. As illustrated in this chapter, this methodology facilitates genetic studies of the human cell surface ranging from the initial chromosome mapping of a surface antigen gene to its isolation and cloning.
The author is indebted to John T. Hart and Donna Babbitt for expert technical assistance and comments on this manuscript.
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