Chapter Thirteen - Gene Trap Mutagenesis in the Mouse
Introduction
The generation of mutations in the mouse is a prerequisite for understanding processes as diverse as development, disease, and cancer, and it is therefore the subject of concerted efforts worldwide to disrupt the function of all mouse genes. Three main strategies have been used to generate mutations: first, gene targeting by homologous recombination is used to produce defined mutations in specific genes; second, point mutations or small deletions can be generated by chemical mutagenesis using agents such as ENU or EMS; and third, random gene trap mutagenesis can be applied genome-wide using plasmid, virus, or transposon DNA vectors. These three approaches create different types of alleles, and they complement each other in the creation of allelic series for all mouse genes. The recent advances in sequencing of mammalian genomes have facilitated the expansion of these approaches to large-scale mutagenesis programs, which aim at generating public resources of mouse mutant alleles (for review, see Gondo, 2008). It will be this collection of allelic variants, with mutations ranging from complete deletion to subtle modifications, which will eventually allow us to dissect gene function at a detailed level.
In this chapter, we review different approaches of gene trapping in mouse embryonic stem (ES) cells, and we provide detailed experimental protocols for trapping by retroviral and transposon vectors. The last section contains a practical guide for the ordering and handling of gene trap clones from consortia. Readers are also referred to Chapter 14 that discusses further aspects of gene trap mutagenesis.
Section snippets
Gene Trapping Strategies
The concept of using insertional mutagenesis to disrupt gene function takes its source from the classic work of Barbara McClintock with transposons in maize. In the mouse, endogenous or exogenous proviruses as well as microinjected DNA can disrupt gene function by insertional mutagenesis (Copeland et al., 1983, Schnieke et al., 1983, Wagner et al., 1983). It was shown that transgene expression can be subject to gene regulatory elements in the vicinity of the insertion site (Jaenisch et al., 1981
Splicing elements
A critical point in design of the gene trap cassette is the choice of splice acceptors and donors. For 5′ splicing in promoter trapping, we recommend the adenoviral major late gene exon 2 splice acceptor (Friedrich and Soriano, 1991). We have not observed by-passing of this splice acceptor, which could potentially result in nonmutagenic or hypomorphic trap insertions, in any of 24 mutant mouse lines that we have analyzed at a molecular level. The adenoviral major late gene exon 2 splice donor
Generation of retroviral vectors
A retroviral gene trap vector is generated by cloning of a plasmid that contains a gene trap cassette into two flanking 5′ and 3′ long terminal repeat (LTR) elements of the Moloney Murine Leukemia Virus (MMLV). The total size of the retroviral vector, including LTRs and trap cassette, should not exceed 11 kb. The direction of the trap cassette should be in reverse to the 5′–3′ orientation of the viral LTRs (Friedrich and Soriano, 1991), and the LTRs should be engineered to lack viral enhancers (
Generation of transposon plasmids
This protocol is described for PiggyBac vectors, but can be easily adapted for the application of the Sleeping Beauty transposon. For gene trapping with a PiggyBac transposon, generate a plasmid that contains a gene trap cassette that is flanked by the 5′ and 3′ PiggyBac terminal repeats (TR). For efficient transposition of PiggyBac, the minimal sizes for the TRs have been determined to be 310 bp for the 5′TR and 235 bp for the 3′TR (these TRs also contain nonrepeating internal sequences that
Identification of Trap Insertion Sites by Splinkerette PCR
The insertion sites of gene traps have been previously mainly determined by methods that generate cDNA fragments containing parts of the vector cassette and exons flanking the trap insertion. For promoter traps, intronic locations can be determined by sequencing exons upstream of the trap insertion with the 5′ RACE method, and for polyA traps, insertions can be identified by 3′ RACE to sequence exons downstream of the insertion. Detailed protocols for 5′ and 3′ RACE analysis of gene trap clones
Ordering and Handling of Gene Trap Clones from Consortia
Collections of gene trap clones have been generated by several laboratories worldwide (see Table 13.1 for web links to gene trap consortia and databases). The annotation data of these clones has been combined in a central database, accessible at the web site of the International Gene Trap Consortium (IGTC; www.igtc.org). Other useful databases for accessing gene trap clones can be found at the UniTrap web site (http://unitrap.cbm.fvg.it), and at the Mouse Genome Informatics web site (//www.informatics.jax.org
Outlook
Gene trapping has been one of the most successful strategies for the mutagenesis of the mammalian genome, and it is a cornerstone in the worldwide efforts to generate a public library of ES cells with mutations in all mouse genes. Although several consortia have shifted their efforts now on gene targeting, which allows the tailored modification of all genes, gene trapping remains by far the most efficient mutagenesis strategy for the generation of large collections of mutant alleles. Powerful
Acknowledgments
We thank our colleagues for critical reading of the manuscript. Work in the authors’ laboratory is supported by grants from the National Institute of Child Health and Human Development to P. S.
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TRACE generates fluorescent human reporter cell lines to characterize epigenetic pathways
2022, Molecular CellCitation Excerpt :Lentiviral integration into a gene might disrupt its function in a manner that impacts the downstream analysis of a pathway of interest. However, the lentiviral vectors used in TRACE do not contain the splice acceptor or donor sequences that feature in the majority of gene-trap vectors (Friedel and Soriano, 2010); hence, we do not expect them to efficiently disrupt gene function unless integrated into an exon. Nevertheless, care should be taken when choosing reporter integration sites for downstream analysis by prioritizing reporters that have integrated in a reverse orientation in a gene, or in intergenic regions of the genome.
Harnessing model organism genomics to underpin the machine learning-based prediction of essential genes in eukaryotes – Biotechnological implications
2022, Biotechnology AdvancesCitation Excerpt :However, a possible limitation might be that haploid cells or homozygous (inbred) organisms need to be used to achieve complete gene inactivation and lethality. A gene trap method that was initially developed for the bacterium Escherichia coli (see Casadaban and Cohen, 1979) was later applied to D. melanogaster (see Bellen, 1999) and to mice (Stanford et al., 2001; Friedel and Soriano, 2010). A similar approach utilising mouse retroviral vectors (Kurian et al., 2000) was applied to the zebrafish, Danio rerio (see Golling et al., 2002), revealing that 315 of its genes were essential for development (Amsterdam et al., 2004).
TAOK3 is a MAP3K contributing to osteoblast differentiation and skeletal mineralization
2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Statistical significance was accepted as a p-value less than 0.05. To evaluate the function of TAOK3 in osteoblasts, mice bearing a homozygous gene trap-mediated loss-of-function Taok3 allele were examined [21]. To validate the efficacy of the Taok3 gene trap allele, calvarial osteoblasts (COBs) from homozygous Taok3 gene trap mice (hereafter, Taok3−/− mice) were subjected to qPCR.
Cre/lox generation of a novel whole-body Kiss1r KO mouse line recapitulates a hypogonadal, obese, and metabolically-impaired phenotype
2019, Molecular and Cellular EndocrinologyLipin-1 and lipin-3 together determine adiposity in vivo
2014, Molecular MetabolismCitation Excerpt :Here, we employed single- and double-knockout models to investigate the possibility that lipin-3 plays an important role in adipose tissue in vivo. Lpin3KO mice were generated by targeted trapping [23]. The trapping vector, which contained a splice acceptor site upstream of a promoterless β-geo cassette, was inserted into intron 3 of Lpin3.
Gene Trapping
2013, Brenner's Encyclopedia of Genetics: Second Edition