Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries ( The 1.7-kb PCR product was subclon...)

Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

The 1.7-kb PCR product was subcloned, sequenced, and found to encode not the IL-4 R but the human IgG FcaRII receptor, a receptor found on most hematopoietic cell types that recognizes the Fc domain of IgG antibodies.¹⁴ Although the a-IL-4 R monoclonal antibody used in the FACS sort was derived from mouse cells, it is not surprising that the human IgG Fc receptor can recognize the Fc region of murine IgG because there is a high degree of homology between both the murine and human IgG constant regions, as well as between their respective IgG FcaRII receptor chains.¹⁴ Thus, the a-IL-4 R antibody functioned in this screen not as an IL-4 R-specific antibody but as an IgG FcaRII-specific ligand. This serves to better illustrate the power of FACS for receptor cloning because, when screening for an unknown receptor, it is more likely to have at hand receptor-specific ligand rather than receptor-specific antibody.

Conclusions

ViraPort retroviral libraries can be used in a wide range of applications and are limited only by the ability to devise a functional screen for the desired target gene. In addition, libraries comprising a complex spectrum of mutants derived from a single gene can be screened for dominant positive or negative mutations or for other altered or enhanced phenotypes.¹ The availability of packaging systems that use polytropic envelope proteins, such as 10A1 and VSV-G, will allow functional screening in essentially any mitotic cell type.² Finally, the relatively high titers and high expression levels that can be attained using the pFB vector backbone⁶ increase the probability of being able to identify the desired cDNA from a complex, fully representative library.

Acknowledgments

The authors thank Dr. Gary P. Nolan (Stanford), Dr. John Wu (Berlex), Mei Vaillancourt (Canji), Dr. Mary Simcox (Hoffman-La Roche),
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( The 1.7-kb PCR product was subclon...)Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

Conclusions

Acknowledgments