Chapter 1
Overview on Mouse Mutagenesis
Ralf Ku¨ hn and Wolfgang Wurst
Abstract
In this chapter we give an overview of mutagenesis methods in the mouse as they evolved over the last two decades, an outlook of ongoing and future developments and advice for choosing a mutagenesis strategy. Where appropriate, reference is given to relevant chapters of this book, key original articles and links of web-based resources for mouse mutagenesis.
Key words: Knockout mice, conditional mutagenesis, CreloxP, RNAi knockdown, ENU mutagenesis, chimaeric mice.
1. Mutagenesis Strategies
Gene Knockout Protocols is considered an information resource for beginners in the field of mouse mutagenesis. This chapter gives a shortreviewofmutagenesismethodsastheyevolvedovertimeand an outlook of the ongoing developments to provide an integrated view of the specialized protocols in this volume.
Strategies for mutagenesis can be classified into the reverse or forward genetics approach. Forward genetics is a phenotype-driven approach whereby large numbers of mutations are induced at ran-dom and new mutants are identified through specific phenotype screens Fig.1.1. In mice, experimentally induced forward genet-ics methods include irradiation, chemical mutagenesis with ethylnitrosourea ENU and transposon-based mutagenesis. Depending on the nature of the mutagen mainly chromosomal aberrations, point mutations or insertions are induced. Since no prior assumption is made about the underlying genes the forward genetics approach represents an unbiased way for the identification
Ralf Ku¨ hn, Wolfgang Wurst eds., Gene Knockout Protocols: Second Edition, vol. 530 aHumana Press, a part of Springer Science+Business Media, LLC 2009 DOI 10.1007978-1-59745-471-1_1 Springerprotocols.com
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Reverse Genetics Forward Genetics
Gene driven Phenotype driven
Gene Targeting ENU in vivo Gene Trap Transposons ENU in vitro Irradiation Knockdown
Phenotyping Gene Identification
Fig.1.1. Comparison of reverse and forward mutagenesis strategies. For the reverse genetics approach a preselected gene is manipulated in vitro in embryonic stem ES cells that are used to generate a mutant mouse strain. The phenotypic characterization of the mutant should reveal the essential, nonredundant function of the targeted gene. By contrast, forward genetics randomly mutagenizes a large number of genes in vivo. Upon breeding of the founder generation relevant mutant offspring are identified by phenotypic screening. The altered gene must be identified by genetic mapping and sequence analysis.
of genes and genetic pathways involved in biological processes. However, since large numbers of mice must be raised and handled over years such screens are usually performed only by large research centres. Besides chapters on transposon mutagenesis Chapter 20 andchemicalmutagenesisinembryonicstemEScellsChapter7, this book also focuses on methods of reverse mouse genetics that are more suited for individual researchers and smaller research units.
The reverse genetics approach requires knowledge about the sequence and structure of a target gene and aims to char-acterize its in vivo function by the generation of a mutant mouse strain, the phenotype of which is compared to wild-type controls. This strategy, which includes the production of knockout mice by gene targeting, gene-trap mutagenesis,
Overview on Mouse Mutagenesis
chemical ES cell mutagenesis and RNA interference RNAi-mediated knockdown Fig. 1.1, relies on the use of murine ES cell lines. ES cell lines exhibit unique properties such as the ability, once established from the inner cell mass of a mouse blastocyst, to renew indefinitively in cell culture while retaining their early pluripotent differentiation state. This property enables to grow ES cells in large numbers and to select, since most mutagenesis methods are inefficient, rare genetic variants that are expanded into a pure stem cell clone that harbours a specific genetic alteration in the target gene Fig. 1.2. Upon introduction of ES cells into mouse blastocysts and embryo transfer these cells contribute to all cell types of the developing chimaeric embryo, including the germline. By mating of germ-line chimaeras to normal mice the engineered genetic modifica-tion is inherited to their offspring and thereby transferred into the mouse germline Fig. 1.2.
The technical basis for reverse mouse genetics was initially established in the decade of 1980?1990 in three steps and the
basic scheme is followed since then, essentially unchanged. The
Wildtype Gene Mutagenic
In vitro
Culture Event Blastocyst ES Cell Culture
Isolation of Recombinant ES Cell Clones
Embryo-
Breeding
Blastocyst
transfer Chimaeric Mouse Establishment of Injection Mutant Strain
Fig. 1.2. Generation of knockout mouse mutants by gene targeting in embryonic stem ES cells. ES cells, initially isolated from mouse blastocysts, can be expanded in vitro to large numbers and used to induce rare genetic variants. Gene-targeting vectors are for homologous recombination with the wild-type gene are used as mutagenic event. A mutant, recombinant ES cell clone must be isolated to generate germline chimaeric mice through microinjection into blastocysts. Further breeding of the chimaeras allows to transfer the mutant allele to the next generation and the establishment of a new mutant strain.
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first of these steps was the establishment of ES cell lines from cultured murine blastocysts and of culture conditions that maintain their pluripotent differentiation state in vitro 1, 2. A few years later it was first reported that ES cells, upon microinjection into blastocysts, are able to colonize the germline in chimaeric mice 3, 4. The third step concerns the technology to introduce preplanned, inactivating mutations into target genes in ES cells by homologous recombination between a gene-targeting vector and endogenous loci. Initially demonstrated for the directly selectable HPRT gene 5, gene targeting was soon adopted for many other genes 6, and the establishment of the first knockout mouse strain was reported in 1989 7. The reverse genetics approach proved very successful and led to the generation of more than 3000 knockout mouse lines that provided a wealth of information on in vivo gene functions. The basic technology for the pro-duction of knockout mice is covered in this book by six chap-ters on gene targeting in ES cells, ES cell manipulation, ES cell line establishment and the production of chimaeras through blastocyst injection Chapters 8?10 and 14?16.
However, even the impressive number of 3000 mutant strains that have been generated within the last two decades represents only a small fraction of all genes contained in the mouse genome. One reason for this situation was the incom-plete knowledge of the mouse genome sequence before 2002. Another reason for slow progress lies in the efforts of 1?2 years of benchwork required to establish a new knockout strain fol-lowing the classical gene-targeting protocols. Therefore, a high demand exists to develop and implement new and more effi-cient procedures for vector construction, ES cell mutagenesis and chimaera production, many of which are included in this volume. Since just the construction of a gene-targeting vector for homologous recombination by PCR and standard cloning methods often requires up to 6 months, streamlined procedures have been developed that allow to assemble such vectors within a matter of days to weeks by homologous recombination in Escherichia coli. Two related protocols, ET cloning 8 and recombineering 9 Chapter 2 that start with genomic BAC clones, were first described in 1999 and 2001 and further developed in later years 10, 11. Since the whole mouse gen-ome is available in the form of sequenced BAC clones all genes are readily accessible to these methods. Besides the more effi-cient construction of gene-targeting vectors, several new strate-gies for mutagenesis in ES cells were added to the basic scheme in later years that do not rely on homologous recombination and do not require the construction of individual gene-targeting vectors.
Overview on Mouse Mutagenesis
Among the four technologies of gene trapping, chemical mutagenesis, oligonucleotide targeting, and RNAi-based knock-down, the first two, gene-trap and chemical mutagenesis, can be performed as a systematic, large-scale process. As described in Chapter3 gene-trap mutagenesis is based on the random integra-tion of an insertion mutagenesis vector across the genome of ES cells and the disruption of trapped genes through vector-specific elements. Gene-trap vectors simultaneously mutate a gene at the siteofinsertion,provideasequencetagfortheidentificationofthe disruptedgene,andindicatetheexpressionofthetaggedgenebya reporter gene. Since a single DNA or retroviral vector can be used to hit a large number of genes, gene trapping is a high-throughput insertional mutagenesis approach that enables to establish libraries of mutant ES cell clones rapidly and at low costs 12. The result-ing databases of mutant genes provide the basis for the establish-ment of mutant mouse strains through germline chimaeras raised from selected ES cell clones. Based on several national gene-trap projects the International Gene Trap Consortium combines the local resources and provides access to a large number of mutant ES cell clones Table 1.1.
A method that does not require the use of vectors for gene mutagenesis in ES cells relies on the use of chemical mutagens like EMS or ENU 13. As described in Chapter 7, a library of mutagenized ES cells is produced in vitro and serves as the basis for the production of germline chimaeras and mutagenized off-spring that are screened for phenotypic alterations. Alternatively, the library of mutagenized ES cells can be screened by RT-PCR and sequencing to identify clones that harbour mutations in a specific gene 14. A collection of mutant clones allows to estab-lish allelic series of mutants and to map functionally important residues of a protein at high resolution. The introduction of a preselected point mutation into a gene, for example, to mimic a human disease allele, has traditionally been a labour-intensive task that requires the construction of a specific gene-targeting vector. However, it has recently been found that synthetic oli-gonucleotides can act in ES cells as targeting vector surrogate if DNA repair mechanisms are transiently suppressed 15.This method of oligonucleotide targeting Chapter 5 simplifies the introduction of point mutations into the genome and greatly facilitates the production of specific disease models. Another recently emerging mouse mutagenesis method is RNAi-mediated gene silencing. RNAi has developed into a routine method to knock down genes in cultured cell lines but has also been found useful for silencing gene expression in embryos and adult mice 16. In this book a method for RNAi mediated gene silencing through shRNA vector transgenesis is described in Chapter 6.
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Table 1.1 Web-based resources for mouse mutagenesis
continued
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