RESTRICTING NUCLEAR PROTEIN TO SPECIFIC PHASES OF THE CELL CYCLE

摘要

The present invention relates generally to mutagenesis of target genes that enhances the natural mutagenic capabilities of adaptive immune cells by providing a chimeric construct that exploits the ability of molecules such as AID to stimulate diversification and the ability of a second molecule to restrict nuclear activity of the molecules and/or protect cell viability. The invention provides a method for stimulating diversification in expressed genes, such as antibody genes, using polypeptides whose nuclear activity is restricted to specific phases of the cell cycle. This method can be coupled with selection to identify B cell clones that produce, for example, antibodies of high affinity or specificity, or for developing T cells for immunotherapy. The invention provides an improved means of developing a repertoire of variant immunoglobulins and other polypeptides.

主权项

1. A nucleic acid construct comprising:
(a) a first nucleotide sequence that expresses an activation-induced cytosine deaminase (AID) polypeptide, wherein the AID polypeptide is modified to prevent nuclear export; and (b) a second nucleotide sequence that expresses chromatin licensing and DNA replication factor 1 (CDT1) or another polypeptide targeted for cell cycle-dependent nuclear destruction, wherein the second nucleotide sequence is operably linked to the first nucleotide sequence.