title: Retroviral vectors to monitor somatic hypermutation.
abstract: The recent expansion of studies on hypermutation may benefit from a fast and uncomplicated way to measure mutation rates. In this paper we compare different retroviral vector designs for monitoring hypermutation in vivo. Retroviral vectors combine a high transduction rate with integration at random sites within the host cell genome, thus equalizing positional effects on the reporter gene. The vectors contain a reporter gene with a premature TAG termination codon; upon reversion, a full-length fluorescent protein is expressed. Any single point mutation at the amber codon activates the reporter--except the transition from G to A, which only creates the stop codon TAA. In the construct, the reporter gene is followed by an internal ribosome entry site and a second marker that allows selection of stably transduced cells. As a reporter gene, we tested the green and yellow fluorescence proteins (GFP and YFP); and various proteins with red fluorescence (dsRed). The second marker was either a drug resistance gene, or a second fluorescent protein. We also introduced various cis-acting enhancer elements into the reporter construct, to study the simultaneous activity of enhancers on transcription and hypermutation. We found that GFP as a reporter, combined with a drug selection marker, gave the most consistent and convenient mutation rate measurements. DsRed is a good alternative to GFP, but variants with greater fluorescence intensity are needed when combined with green fluorescence measurements. We also confirm that no immunoglobulin specific sequence is needed to target hypermutation. Depending on their position in these ectopically expressed constructs, enhancers can have positive or negative effects on hypermutation.
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0670, USA.
authors.forename: Maik
authors.initials: M
authors.lastname: Klasen
authors.forename: Freia J X
authors.initials: FJ
authors.lastname: Spillmann
authors.forename: James B
authors.initials: JB
authors.lastname: Lorens
authors.forename: Matthias
authors.initials: M
authors.lastname: Wabl
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018388:Codon, Terminator / Q000235:genetics; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics*; D027041:Somatic Hypermutation, Immunoglobulin; D014161:Transduction, Genetic
chemical_list: D018388:Codon, Terminator; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D012194:RNA-Directed DNA Polymerase