Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414, USA
1 To whom correspondence should be addressed. E-mail: cliffw{at}itsa.ucsf.edu
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Abstract |
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Keywords: B cell/hypermutation/mutagenesis
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Introduction |
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To direct more efficiently the evolution of proteins and other biological products, researchers have sought better ways to introduce mutations. An organism can be exposed to a mutagen, e.g. nitrosoguanidine or UV light, and as a result mutations occur at a higher rate. A disadvantage is that mutagens not only mutate the desired genes but also can indiscriminately mutate genes necessary for survival of the organism. As a result, the toxicity of the mutagen may cripple the organism before enough iterations of mutation and selection can evolve a desired product.
To minimize injury to the organism, mutagenesis of target genes can be performed in vitro (Stemmer, 1994; Ness et al., 1999
; Schmidt-Dannert et al., 2000
). Although this has proved to be a good tool to improve proteins, the genetic diversity that can be accessed is still limited. While the polymerase chain reaction (PCR) techniques available may or may not be a limiting factor, the other steps are equally important in the process. The size of a gene library is limited by the plasmid cloning efficiency and the number of genes that can be screened is limited by the transformation efficiency of Escherichia coli or other host. It may seem that current technology can effectively handle these tasks and that the ceiling for genetic diversity is fairly high. Yet because of the exceedingly many combinations in which amino acids can be joined, laboratory bench manipulations invariably do limit the scope of mutagenesis.
In cases where in vitro mutagenesis is not ideal, mutagenesis of desired genes might be better performed in vivo. An organism would produce its own mutagen, yet be minimally harmed by it. In nature, the prime example of such a scheme is the creation of antibodies. The genetic diversity created by the immune system is so large that for nearly every foreign antigen, an antibody can be generated to bind it. After combinatorial rearrangement of gene segments, the antibody variable region is mutated at a rate orders of magnitude greater than the spontaneous mutation rate. This occurs when activated B cells encounter antigen and is termed immunoglobulin (Ig) somatic hypermutation (Weigert et al., 1970; Kim et al., 1981
; McKean et al., 1984
). Somatic hypermutation requires activation-induced cytidine deaminase (AID) (Muramatsu et al., 2000
). Although the exact physiological role for AID remains unclear (Doi et al., 2003
; Ito et al., 2004
), it is believed that AID deaminates deoxycytidine residues (Bransteitter et al., 2003
; Pham et al., 2003
) and that point mutations are introduced when DNA repair mechanisms attempt to rectify the AID-mediated DNA damage (Cascalho et al., 1998
; Di Noia and Neuberger, 2002
; Petersen-Mahrt et al., 2002
; Neuberger et al., 2003
).
It is remarkable that activated B cells can mutate at such a high rate so as to improve the antibody repertoire, yet mutate in such a way that cells can survive and proliferate. It was long believed that activated B cells hypermutated only the variable region and that Ig gene sequences directed the mutating factors to its target (Betz et al., 1994; Bachl and Wabl, 1996
; Bachl et al., 1998
). Along these lines, it was shown that a transformed B cell line can hypermutate its endogenous Ig variable region and be selected for antibodies with increased binding to streptavidin (Cumbers et al., 2002
). Recently, it has been shown that Ig gene sequences are not necessary for hypermutation (Shen et al., 1998
; Gordon et al., 2003
; Wang et al., 2004
). Here we investigated this mechanism as a general mutagenesis tool, one not limited to antibodies.
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Materials and methods |
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Constructs were based on the MoMLV retroviral vector contained in p102.21 (kindly provided by J.B.Lorens, Rigel Pharma, South San Francisco). They contain the internal ribosome entry site (IRES) and puromycin resistance gene of pIRESpuro3 (Clontech). The positive control vector, pEGFP-Ipuro, contained EGFP from pEGFP-N1 (Clontech).
To create the hypermutation substrate, EGFPevo, two nucleotides of EGFP were substituted. The first substitution, C197 to T, changed Thr66 to Ile. Although the second substitution, C198 to A, resulted in no amino acid change, it helped to distinguish true hypermutation revertants (EGFPevo T197C) from potential EGFP contaminants that could be introduced during cell sorting, PCR amplification or other manipulations. EGFPevo was inserted into pEGFP-Ipuro in place of EGFP to create pEGFPevo-Ipuro.
Cell culture
Retroviral vectors were packaged by PhoenixEco cells (ATCC SD 3444). The 1881, 70Z/3 and MPC-11 cells were infected with the vectors and selected in the presence of 2.5 µg/ml puromycin for 7 days. Our procedure infected 5% of the cells; hence by assuming a Poisson distribution for the multiplicity of infection, 95% of transduced cells will contain only one copy of the transgene. In order to start experiments with zero fluorescent mutants, fluorescent cells were removed by FACS (fluorescence-activated cell sorting). After FACS, cells were grown with 1.5 µg/ml puromycin and were passaged daily. Cultures contained a total of 21 ml and each day 14 ml of cells were removed and replenished with 14 ml of fresh medium. Each culture contained
63 x 106 total cells at the time of passaging, with 21 x 106 retained daily. Fluorescent cells were detected by flow cytometry. Single cells were isolated by FACS and clones were expanded from single cells. The 1881, 70Z/3 and MPC-11 cultures were grown in RPMI 1640 media with 10% FCS.
Immunoblotting
Whole cell extracts were performed using standard methods. Cells were incubated in lysis buffer [0.5% Triton X-100, 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.2 mM PMSF, 0.2 µg/ml leupeptin, 0.75 µg/ml pepstatin A, 10 µg/ml aprotinin] and the soluble fraction was separated by SDSPAGE using a 12% acrylamide gel. Electrophoresed proteins were transferred by Western blotting onto a nitrocellulose membrane. Specific proteins were probed using a rabbit anti-GFP antibody (Masaki Fukata) and a mouse monoclonal anti-actin antibody (Calbiochem). Blots were incubated with horseradish peroxidase-coupled secondary antibodies and visualized using the Amersham enhanced-chemiluminiscence (ECL) method.
DNA sequencing
DNA from the 1881 clones was isolated and the GFPevo gene was PCR amplified using Pfu polymerase (Stratagene). The PCR products were incubated with Taq polymerase to add deoxyadenosine overhangs and then cloned into pCR2.1-TOPO (Invitrogen). The plasmids were then amplified in E.coli and sequenced.
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Results and discussion |
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After only 5 days, fluorescent cells were detected and after 32 days several mutants (1.4 x 105) with varying fluorescence intensities were observed (Figure 1C). A fraction of cells exhibited a high level of fluorescence whereas the majority fluoresced with intermediate intensity. Fluorescent cells were isolated by fluorescence-activated cell sorting (FACS) and clones were expanded from single cells. DNA sequencing of the EGFPevo substrate in these clones revealed various mutations including missense, nonsense and silent mutations (Figure 2, Table I). Proteins were extracted from several clones and immunoblotted (Figure 3) using antibodies against EGFP and actin, which served as a reference protein. The amounts of EGFPevo were of the same order and any differences from clone to clone could not account for the 68885-fold increases in fluorescence observed in these clones expressing mutated EGFPevo. This strongly suggested that the mutations in EGFPevo led not to increases in protein production, but to structural improvements that increased the fluorescence of the molecule.
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When we expressed the EGFP vectors in cell lines of B lineage that do not express AID, 70Z/3 and MPC-11, zero mutations were found after 32 days (not shown). Almost certainly, application of B cell hypermutation as a mutagenesis tool will require cells that produce the AID mutagen. It has recently been shown that hypermutation can occur not only in lymphocytes, but also in fibroblasts producing transgenic AID (Yoshikawa et al., 2002). Hence it is possible that any cell line made to produce AID could serve to mutate exogenous genes. However, recent studies suggest that other factors interact with AID (Honjo, 2002
; Ta et al., 2003
; Ito et al., 2004
) and cells of B cell lineage that produce such factors in addition to AID may be optimal for mutagenesis applications. The 1881 cell line is a mouse cell line of B cell lineage that produces AID endogenously (Bachl et al., 2001
). Like activated B cells, it divides quickly (5 h minimum doubling time), hypermutates the endogenous Ig variable region and undergoes Ig isotype class switching (Wabl et al., 1985
; Meyer et al., 1986
; Jack et al., 1988
). Furthermore, 1881 (or other activated B cell lines) may be suitable for in vivo mutagenesis because they not only produce components necessary for hypermutation, but also are adapted to and tolerate the mutagen.
Retroviral gene delivery can efficiently access hypermutating sites
Previous results have suggested that only a fraction of locations throughout the genome can mutate at a high rate (Wang et al., 2004). It also has been found that many housekeeping genes in activated B cells are not hypermutated (Storb et al., 1998
) and it is perhaps differences in chromatin structure (Woo et al., 2003
) that determine what locations of the genome do or do not hypermutate. To access the hypermutating locations, in the past we and others have transduced plasmid reporter constructs and screened for clones that can hypermutate (Bachl and Wabl, 1996
; Bachl et al., 1998
, 2001
). This approach proved too labor intensive for any engineering application and sometimes even made experimental results difficult to interpret. To develop a practical method for mutagenesis, it was necessary to use a more efficient means of gene delivery. Retroviral infection was well suited for this application because an exogenous gene can be delivered to many cells in a single step. In our experiments we stably transduced 5% of a culture containing 106 cells, thus producing
50 000 transduced cells at one time. Because the site of gene integration by retroviral infection is essentially random, among the many different sites accessed in one infection step, a fraction is almost certain to support hypermutation.
Hypermutation could be a platform for in vivo directed evolution
The fluorescence-restoring mutations that we identified would probably have been difficult to predict ab initio and site-directed (non-random) mutagenesis of EGFPevo would have been cumbersome. We instead identified the mutations in a single experiment where continual mutagenesis was performed merely by keeping the infected 1881 cells in culture. While application of an external mutagen to other cells could harm them, the 1881 cell line naturally produces its own mutagen without noticable ill-effects. Although certain strains of yeast or bacteria also might be used for in vivo mutagenesis, the 1881 cell or other AID-producing cell lines may be particularly useful for proteins that need to be produced in mammalian cells. Previous studies have shown that AID-producing cell lines can create mutations in transgenes such as neomycin phosphotransferase (Yelamos et al., 1995) or EGFP (Yelamos et al., 1995
; Bachl et al., 2001
; Yoshikawa et al., 2002
; Wang et al., 2004
). In these studies using EGFP, a premature stop codon was inserted into the gene so that reversion of the stop codon would restore fluorescence of the protein; the gene was used as a reporter to measure mutation rates. In this reporter gene, mutations at the stop codon were expected because the stop codon was engineered into an RGYW (R = A or G, Y = C or T, W = A or T) motif (Rogozin and Kolchanov, 1992
), a site with a high likelihood of cytidine deaminase activity and hypermutation. Here we showed that hypermutation of a retroviral substrate can create unexpected mutations that give rise to a desired phenotype. This demonstrates that the scope (i.e. gene coverage) and rate of mutagenesis are sufficient for a directed evolution application. A future goal should be to engineer cultures that not only mutate exogenous genes, but also self-select for mutants with desired traits. Towards this end, utilization of the components that generate antibody diversity could provide a powerful platform for mutagenesis.
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Acknowledgments |
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References |
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Received July 20, 2004; revised October 4, 2004; accepted October 11, 2004.
Edited by Paul Carter
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