Protein evolution by hypermutation and selection in the B cell line DT40

Abstract

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible.

A Dendritic Guidance Receptor Complex Brings Together Distinct Actin Regulators to Drive Efficient F-Actin Assembly and Branching

“…Briefly, worms expressing an integrated PVD>dma-1::GFPnovo2 marker (with the GFPnovo2 inserted before the cytosolic domain of DMA-1 to avoid hindering the PDZ binding motif) (Arakawa et al, 2008) were injected with extrachromosomal arrays carrying PVD>hpo-30::mcherry , PVD>K10D6.2::mcherry or PVD>clc-1::mcherry . Expression and membrane localization of the proteins were verified using regular fluorescent microscopy.…”

A double-strand break can trigger immunoglobulin gene conversion

“…Integration of the I-SceI reporter vector into the rearranged Ig light chain locus was screened using the PS148/VL533 primer pairs and was further verified by PCR amplification of the VJ intervening sequence of the un-rearranged locus using the VL513/VL514 primer pairs as previously described (36). AID −/− IgL Sce GFP was transfected with a Tet-On 3G expression vector, pChr2RsvProTetOn3gPuro and integration of the vector into the target locus was screened using the CHR0208/RS16 primer pairs.…”

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

“…B cell lymphoma lines with misregulated AID-dependent somatic hypermutation have been used to evolve antibodies or fluorescent proteins (Arakawa et al, 2008; Lim et al, 2016; Wang et al, 2004), but the promiscuous mutagenesis in this system is relatively inefficient. In contrast, by directly recruiting AID, diversifying base editing systems (CRISPR-X and TAM) can facilitate efficient, targeted mutagenesis of key structural or functional domains of endogenous targets through carefully designed sgRNA pools.…”