Site-directed and random mutagenesis is an indispensable tool in studying the structures and functions of proteins including those that are critical in drug addiction. However, the in vitro mutagenesis procedures are very labor intensive and of low throughput. Somatic hypermutation (SHM), one of the main mechanisms that generate the great diversity of antibodies in B lymphocytes, can be used to randomly mutate exogenous Genes Of Interest (GOI) that encode non-antibody proteins. Since mutations occur independently in each cell, millions of random mutants are easily generated and can be screened quickly when a proper selection method is set up. The SHM rate is much higher (up to 106 fold) at certain loci (such as loci for immunoglobulin V regions) than the rest of the cell genome. However, there is no convenient way to insert the GOI at loci of high SHM. Targeted homologous recombination can not be performed because the sequences at these loci are heterogeneous and different from cell to cell. Therefore, we propose to generate novel B-lymphoma cell lines that allow easy insertion of a GOI to loci of high SHM. Millions of mutants can be generated and expressed in the cell lines. Certain properties of the GOI mutants can be screened and selected for further studies. The mutagenesis procedure can be repeated multiple times to evolve the GOI to desired properties. We will generate the cell lines by integrating a DNA construct into the cell genome randomly that contains an insertion guide marker and a mutation reporter gene. We will perform a prescreening and two rounds of screening to isolate cells with the reporter gene mutated in a single cell division. Such cells should have the reporter gene integrated at loci of very high SHM rates. The insertion guide marker will allow easy and specific insertion of a GOI at the high SHM loci. In addition, the GOI will be controlled by an inducible promoter and thus SHM can be turned up and down because SHM rates are proportional to the levels of gene expression. In future studies, we plan to use this system to screen for mutants of the monoamine transporters that are insensitive to addictive drugs such as cocaine, amphetamine, methamphetamine, MDMA (ecstacy) or therapeutic drugs, such as Ritalin and Prozac. The cell lines can be used to study many other proteins on their structures, functions, drug binding sites, regulations, etc.
