Elsevier

Methods in Enzymology

Volume 477, 2010, Pages 243-269
Methods in Enzymology

Chapter Thirteen - Gene Trap Mutagenesis in the Mouse

https://doi.org/10.1016/S0076-6879(10)77013-0Get rights and content

Abstract

Gene trapping in mouse embryonic stem (ES) cells is an efficient method for the mutagenesis of the mammalian genome. Insertion of a gene trap vector disrupts gene function, reports gene expression, and provides a convenient tag for the identification of the insertion site. The trap vector can be delivered to ES cells by electroporation of a plasmid, by retroviral infection, or by transposon-mediated insertion. Recent developments in trapping technology involve the utilization of site-specific recombination sites, which allow the induced modification of trap alleles in vitro and in vivo. Gene trapping strategies have also been successfully developed to screen for genes that are acting in specific biological pathways. In this chapter, we review different applications of gene trapping, and we provide detailed experimental protocols for gene trapping in ES cells by retroviral and transposon gene trap vectors.

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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