- 作者: Ming Jie Zhang; Andrew I. Dayton
- 作者服務機構: Laboratory of Molecular Virology, Division of Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Md., USA
- 中文摘要: --
- 英文摘要: We have developed a method for nuclear export signal trapping (NEST) to isolate functional Rev clones from various types of libraries such as libraries of Rev mutants. The expression libraries are cotransfected into COS cells together with a novel Rev-dependent immunoselectable CD28 expression plasmid, pCMV128-CD28. CD28-positive cells are recovered by FACS or by immune precipitation with magnetic beads, and the low-molecular-weight extra chromosomal DNA is recovered, amplified for Rev-containing DNA by PCR and recloned into expression plasmids. The resulting clones are enriched for functional Rev clones. These can be recovered efficiently after several repetitive NEST cycles. This technique may be usefully applied to study various regions of Rev, such as the RNA binding domain and the nuclear export signal, or effector domain and potentially to the isolation of cellular factors with nuclear export capabilities.
- 中文關鍵字: --
- 英文關鍵字: Rev; Nuclear export ; Nucleocytoplasmic transport ; Nuclear export signal