A powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning.
cDNA expression cloning using retroviral vectors provides a means of stably introducing genes into target cells at efficiencies that surpass those achieved by transfection. Furthermore, retroviral vectors allow for the introduction and expression of complex cDNA libraries in a wide range of cell types, including cells of hemopoietic origin. Here we report a novel method for rapidly isolating genes encoding cell surface molecules (CSM) from a human bone marrow stromal cell cDNA library constructed in the retroviral vector, pRUFneo. With a newly described, highly efficient selection strategy using mAb and Ab-coated magnetic beads, we have successfully isolated six cDNA encoding previously defined CSM, including beta 1 integrin and endoglin. Moreover, we have used this approach to define the gene and hence the CSM identified by three previously unclustered mAb. These results confirm previous studies demonstrating the general utility of retroviral cDNA libraries and further extend their use to the expression cloning of cDNA encoding CSM.
Hanson Centre for Cancer Research, Matthew Roberts Laboratory, Division of Haematology, Adelaide, South Australia, Australia.
A C
AC
Zannettino
J R
JR
Rayner
L K
LK
Ashman
T J
TJ
Gonda
P J
PJ
Simmons
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D001854:Bone Marrow Cells; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003238:Connective Tissue / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D015723:Gene Library; D005796:Genes; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells; D006801:Humans; D018189:Immunomagnetic Separation; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012190:Retroviridae / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins