Abstract
Abstract Activation-induced cytidine deaminase (AID) is an essential enzyme for immunoglobulin somatic hypermutation, gene conversion, and class switch recombination in immune cells. Somatic hypermutation is induced by AID mediated cytidine deamination around the V regions of immunoglobulin genes in germinal center B cells. It has been shown that AID also can induce somatic mutations outside Ig loci and in non-lymphoid cells. Our present study attempts to use AID as a target specific mutagen. We propose to confine the mutagenesis function of AID in certain DNA regions by fusing AID with a specific DNA binding protein. We choose the Cre recombinase as the DNA binding protein in the test system. It is hypothesized that Cre-AID fusion protein should promote AID mediated mutagenesis around the loxp site, the binding site of Cre. A reporter system was built by placing a single loxp site next to a GFP expression cassette, which contains stop condon in the middle of GFP coding sequence. If the stop codon is reversely mutated into non-stop codon by AID, GFP signal can be detected. This reporter system would allow us to evaluate whether Cre mediated DNA binding could enhance AID mediated mutagenesis around the loxp site. This experimental strategy has the potential to be used in the directed mutagenesis when Cre is further modified to inactivate the recombinase activity but retain loxp DNA binding activity.
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