2.2.1 Comparative Genomics
The human nuclear genome comprises 23 chromosomes; 22 autosomes and the sex chromosomes, X and Y. The mouse has 20 chromosomes with 19 autosomes. The former has been sequenced to over 90% (as of June 2002, http://genome.ucsc.edu/), and the draft sequence for the mouse has been published (Waterston et al. 2002);
http://www.ensembl.org/Mus_musculus/). The presence of several pairs of homolo-gous genes on one given chromosome defines conserved synteny. The most striking example of conserved synteny is the conservation of gene content of mammalian X chromosomes. In any chromosomal segment of conserved synteny the local gene order is usually conserved to a high degree, making it possible to use mapping information from one species to identify a disease gene in the other species.
2.2.2 Classical mouse mutants and positional cloning
Classical mouse genetics relied on spontaneous or radiation-induced mutations (Lyon and Searle 1989) which in most cases lead to recessive ‘loss-of-function’ phenotypes, but also included cases of ‘gain of function’, i.e. dominant expression of the mutant phenotype. Radiation-induced mutations are often deletions whereas spontaneous mutations include point mutations, retroposon insertions and chromosomal rearrangements. These ‘classical’ mutations certainly represent a selective bias: lethal phenotypes early in life were likely to be overlooked, as were phenotypes which were too mild. Mutant loci were mapped using meiotic segregation in relation to markers of known position. In the early days, these were genes affecting the external appearance like coat color, or allelic forms of enzymes; in recent years, due to rapid PCR tech-niques, these have been replaced by DNA microsatellite polymorphisms. In order to identify the gene the tedious procedure of positional cloning, i.e. high resolution mapping followed by sequencing of sets of overlapping genomic DNA clones is neces-sary unless there is a physiological hint that allows one to focus on a candidate gene.
The availability of a full mouse genome sequence (Waterston et al. 2002) will make this procedure much faster and easier.
In human neurological diseases such as nervus opticus atrophy and several muscle diseases, defects in mitochondrial genes play an important role. We are not aware of reports on spontaneously arising mitochondrial mutations that would cause neurolog-ical diseases in the mouse. However, methods to specifneurolog-ically introduce mutations into the mitochondrial genome have recently been developed (Wallace et al. 2001).
Methods to artificially induce mutations, either random or directed (Fig. 2.1) will be described in the following sections.
2.2.3 Induced random mutations: The ENU screening projects In the course of recent genome projects the chemical mutagenesis of the mouse had an unexpected comeback. Male mice are treated with a sublethal dosage of ethylnitrosurea (ENU), one of the most powerful chemical mutagens. ENU predominantly causes single basepair exchanges (Popp et al. 1983). The offspring of ENU treated males carry multiple paternally inherited point mutations (Balling 2001). To detect neuro-logical mutations in subsequent generations several screening protocols have been set up, such as behavioural, anxiety and pain tests. The common aim of these muta-genesis programs is to saturate the genome with an unbiased spectrum of mutants.
Mutant phenotypes are described in publicly available databases (Germany:
www.gsf.de/ieg/groups/enu-mouse.html; UK: www.mgu.har.mrc.ac.uk/mutabase/;
Fig. 2.1 Origin of mutant mice: Spontaneous and induced mutations. From left to right the methods used to obtain mouse mutants become increasingly sophisticated.
In radiation or chemically induced mutagenesis whole animals are subjected to the treatment by shotgun methods. Using DNA as a mutagen, one chooses the gene to be introduced, but its insertion may be random (as in transgenes) and its nature may be that of a reporter (as in the ‘gene trap’). Whereas transgenes are produced in zygotes, the other manipulations use embryonic stem (ES) cell lines as recipients. In knock-out (KO) and knock-in (KI) technologies, a specific gene is being functionally eliminated or replaced by a variant of interest (usually a human ortholog with a pathogenic mutation). Tissue specific and
temporally controlled gene elimination is achieved by crossing mice with ‘sensitized’ (‘floxed’) genes to those carrying a ‘destructive’ Cre recombinase transgene with a cell type specific and/or inducible promoter.
physical and chemical mutagens random
mating foster mother
chimera
mutant mouse
zygote ES cells, homologous
recombination screen in culture spontaneous
radiation etc. pronucleus injection gene trapping knock out (KO) knock in (KI) ENU
F1: dominant F2/F3: recessive
F1: heterozygous KO/KI F2/F3: homozygous KO/KI F1: transgenic mouse
gene specific, site random
random gene and
site specific DNA mutagenesis
Japan: www.gsc.riken.go.jp/Mouse/; USA: www.jax.org/nmf/documents/about.html;
www.tnmouse.org/).
2.2.4 Transgenes
In transgenic animals genes of interest are added to the genome of a recipient animal.
Expression plasmids (capacity <20 Kb), which in the simplest case consist of a cloned cDNA downstream to a non-specific promoter, are injected into the pronucleus of a zygote. The zygotes are subsequently transferred into the genital tract of foster mothers.
In about ten percent of the injected zygotes the plasmid is integrated into the genome and transgenic offspring are born. The site of transgene insertion is more or less random and usually multiple copies of the transgene are integrated in tandem arrangement. The expression of a transgene depends not only on the promoter used for the construct, but also on the copy number of insertions and on the location of the insertion. For this rea-son several transgenic lines usually have to be analyzed in parallel. To minimize the influ-ence of the genetic environment on a given transgene it is preferable to insert the transgene including its normal chromosomal environment, in the form of a large genomic DNA-fragment (up to several hundred Kb). Yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) have been successfully used for this purpose.
2.2.5 Gene trapping—random mutagenesis of embryonic stem cells
Gene trapping is a random insertional mutagenesis method applied to embryonic stem (ES) cells. The approach is based on the insertion of a promoterless reporter gene, usually the E. coli LacZ gene coding for β-galactosidase. The expression of the reporter, being under transcriptional control of the unknown gene into which it has been inserted, mimicks and allows visualization of the expression pattern of the ‘trapped’
gene. This facilitates the screening for genes of interest. Screening can be performed on the ES cell level for genes active at that stage or — with more relevance to neuro-biology — after induced differentiation of ES cells in culture. Selected ES cell clones are transferred into blastocysts, and may be screened in the resulting chimeras. If they participate in the germ line heterozygous offspring may be used for the analysis of the expression pattern in situ and homozygous mutants obtained in the following genera-tion, in which the insertion would destroy the function of both alleles, are tested for aberrant phenotypes (review: Hill and Wurst 1993).
Skarnes et al. (1995) developed a gene-trapping approach (secretion gene trap) that allows a pre-screen of gene loci encoding membrane and secreted proteins. The vector pGT1.8tm carries, from 5′to 3′, the engrailed 2 splice acceptor, the CD4 trans-membrane domain and a β-geo (lacZ-neoRfusion) sequence (Skarnes et al., 1995).
When insertions occur into genes, the transcripts of which lack a 5′secretion signal sequence, the CD4 transmembrane domain acts as a secretion signal, leading to translocation of the carboxy-terminal end of the translation product into the
endoplasmic reticulum. In the lumen of the ER, the fusion protein is exposed to conditions, including glycosylation enzymes, which lead to inactivation of β-galactosidase activity. However, insertions into a gene encoding a signal sequence, when they occur 3′to the signal sequence, lead to the CD4 domain acting as a simple Type I transmembrane domain, terminating further translocation into the ER. This leaves the 3′transgene product outside of the lumen of the ER in the cytoplasm, providing an active β-galactosidase marker.
2.2.6 Site-specific deletion mutagenesis
Any point mutation, spontaneous or ENU induced, may be combined with targeted deletion mutagenesis. Two different strategies have been developed for targeted dele-tion mutagenesis in mouse ES cells. The first is based on the targeted inserdele-tion of two loxP sites (see 2.2.9) into a specific mouse chromosome by homologous recombination in ES cells, followed by Cre recombinase mediated deletion of the DNA segment between the loxP sites (Ramirez-Solis et al. 1995). ES cell clones transmitted to the germ line yield hemizygous ‘deletion mice’. The second approach utilizes a viral thymi-dine kinase as a negative selection marker, which is introduced into the genomic area of interest by homologous recombination in ES cells. Thereafter ES cells are subjected to X-irradiation induced deletion mutagenesis followed by selection for the loss of thymidine kinase (You et al. 1997). Deletions within the target region are mapped and hemizygous deletion mice are generated.
Hemizygous deletion females are mated with ENU-treated males and the F1 offspring are screened for aberrant phenotypes. Above a background of dominant gain-of-function mutations located anywhere in the mouse genome this approach allows the F1 screening for recessive mutations located within the hemizygous ‘deletion window’ (www.mouse-genome.bcm.tmc.edu/ENU/MutagenesisProj.asp).
2.2.7 Knock-outs: Artificial loss-of-function mutations of known genes
The targeted generation of null-mutations in mice (Thomas and Capecchi 1987) is based on the alteration of a known gene locus by homologous recombination in ES cells. The homologous recombination is driven by a targeting vector which comprises a selection marker (drug resistance) flanked by genomic gene-specific targeting fragments. The targeting vector is transfected into the ES cells via electroporation, resulting clones are selected for drug resistance and screened for the gene-specific homologous recombination event by genomic PCR or Southern blotting. Selected ES cells are injected into blastocysts or aggregated with morulae to yield chimeric blasto-cysts in culture that are subjected to the procedures described for gene trapping (Fig. 2.1). Fifty percent of the ES cell offspring should be heterozygous for the knock-out. Intercrossing yields 25% of homozygous ‘KO mice’ for phenotype analysis. Several thousands of genes have been knocked out and their phenotypes have been character-ized (tbase.jax.org/). The knock-out strategy is not restricted to proper genes, but
might also be useful for the functional characterization of regulatory sequence elements and conserved non-coding sequences, the latter of which have been identified via interspecies sequence comparison (Loots et al. 2000).
2.2.8 Knock-ins of human ‘pathogenes’
In most cases of knock-out experiments a reporter and/or a selection gene is intro-duced concomitantly with disruption of the target gene. The knock-in of a ‘reporter gene’ like LacZ or jellyfish green fluorescent protein (GFP) and its artificial variants allows, if the promoterless reporter is correctly controlled by the target gene regulatory sequences, the analysis of the expression pattern of the target gene in situ. Apart from this somewhat artificial analytical tool, knock-in technology is used to produce accurate models of human disease. A mouse gene can be replaced by a pathogenic allele of the orthologous human gene. Alternatively an equivalent mutation may be introduced into the mouse gene by in vitro mutagenesis and knock-in technology.
2.2.9 Conditional mutations
The value of the knock-out approach for the analysis of complex physiological and behavioural capabilities of an organism has been questioned (Routtenberg 1995). A gene function represents just a node in a complicated network to which other gene functions contribute, modulated by their regulation as well as exogenous influences. If one gene function is missing during the development of an organism, the whole network might react and even compensate for the loss. Thus, the case is certainly different from a ‘minus one’ music recording. This thought was already implicit in Richardt Goldschmidt’s ‘theory of gene physiology’ (Goldschmidt 1927) long before DNA had been identified as the hereditary material. It not only implies that the loss of a seemingly important gene function may cause no overt abnormalities (“no pheno-type”) but also that a single gene defect may change the expression of a host of other genes. Another complication of constitutive gene disruption is the fact that during development one and the same gene may be used at different times in different tissues in a different context. The phenotype resulting from its loss may thus be a super-position of physiologically unrelated events.
In order to avoid these complications, more specific gene targetting methods have been developed. They would either eliminate a gene function specifically in one organ or cell type or at a chosen time, or both.
For tissue specific knock-out, targeted mutagenesis in ES cells has to be silent and should not impair gene function. Usually essential exons are ‘floxed’ by flanking them with loxP sites (short oriented recognition sequences for the phage P1-derived Cre recombinase). Using homologous recombination in ES cells, the floxed exon including a floxed selection marker is introduced into the gene of interest. Targeted ES cell clones are selected and transiently transfected with a Cre-recombinase expression plasmid.
Cre recombinase deletes the sequences between two tandemly arranged loxP sites and
ES cell clones have to be selected which have lost the selection cassette but not the essential exon. ES cell clones with the floxed essential exon are used to obtain homozygous floxed mice as described for conventional knock-outs. These mice are bred with Cre recombinase transgenic mice, the transgene of which is regulated by a tissue specific promoter. In Cre-transgenic homozygously floxed mice the essential exon is deleted exclusively in cells which express the Cre and the effects of the gene knock-out in specific tissues and organs can be analyzed.
Inducible knock-outs utilize “on/off ” promoter systems for the expression of Cre-recombinase transgenes, which can be regulated externally e.g. by application of effector molecules like tetracyclin or steroid hormones, depending on the artificially introduced regulator system. In this case the Cre-mediated deletion of an essential exon of a gene can be induced at a specific timepoint so that critical developmental stages are overcome and acute effects of the loss of function may be studied: see e.g.
the experiments of Malleret et al. (2001) and Gross et al. (2002) using tetracycline regulated systems.