Gene trap mutagenesis

To study the role of one mutation in the pathogenesis of any human disease, or to determine the function of one unknown gene several ways can be followed.

Mutagenesis represents a frequently used method to achieve this purpose. This

20 process can be defined as the modification of the genomic DNA of the organism in a stable way to get a mutation, which was described for the first time more than 80 years ago (Muller 1927).

Old methods to induce mutagenesis included using X-ray radiation or applying chemical mutagenesis by chlorambucil, but these strategies have the problem that several genes can be affected, which makes studying one single gene or mutation so difficult (Russell et al. 1989; Stanford et al. 2001). In contrast with previous methods, ethylnitrosourea (ENU) can cause point mutations or small deletions (20–50 bp) in spermatogonial stem cells, but it has the disadvantage that it gives no landmark for identifying the mutated genes (Russell et al. 1979; Stanford et al. 2001). The first report about using retrovirus to introduce exogenous DNA into the mouse germ cells was almost 40 years ago, and it was possible using this method to recover the affected genes, opening the road for the wide use of insertional mutagenesis (Jaenisch 1976; Spence et al. 1989). The first transgenic mouse was produced in 1981 by the microinjection of DNA into fertilized oocytes (Gordon and Ruddle 1981;

Wagner et al. 1981), but identifying the affected gene after that was still not easy, which made the strategy of homologous recombination in embryonic stem cells (ES) preferred in order to mutate a specific gene (Stanford et al. 2001).

1.5.2 Gene trapping

This strategy can help to produce embryonic stem cells (ES) with random mutations by the inserting of a trapping vector into the genomic DNA using electroporation or retroviral infection. The reporter after the splicing acceptor inside the trapping vector gives a signal indicating its presence in a transcriptionally active gene, and mutating thereby this gene by disrupting the splicing process, taking advantage of the polyA tail after the reporter. The trapped gene can be identified by sequencing the mRNA product using a technique named as rapid amplification of cDNA ends and primers located in the trapping cassette. The trapping vector may not be completely successful to inactivate the affected gene and hypomorphic allele, rather than a null allele, can be generated due to the occurrence of alternative splicing, especially when the trapping vector is inserted into an intron. Different vectors can be used, with variable characteristics and efficacy, which can be classified basically into 3 types (Stanford et al. 2001; http://www.genetrap.org/tutorials/overview.html).

21 The enhancer-trap vector includes inducible minimum promoter that needs to be inserted in an intronic region near to a cis-acting enhancer element (the enhancer of affected gene), which derives the expression of the reporter gene β-galactosidase (LacZ). It makes the affected gene usually hypomorphic, which made it not frequently used. The gene-trap vector produces a fusion transcript between the upstream exon of the mutated gene and the promoterless reporter LacZ by the insertion of a trapping cassette that contains a splice acceptor (SA) upstream of LacZ in an intronic region also. In contrast to that, the promoter-trap vector is inserted into an exon of the affected gene producing thereby a fusion protein with the LacZ reporter. It should be always kept in mind that the mutated protein generated by these different vectors may still be functional depending on the location of its domains (Stanford et al. 2001).

1.5.3 The ‘Knockout-first’ strategy

Several modifications can be applied on the previous trapping vectors, such as the integration of homologous recombination sites, facilitating thereby specific genes to be targeted. The mutated allele can also be further altered so that it can be reverted back to wild type phenotype then reverted again to the null allele-state using different systems, such as Cre- and FLP-Recombinase systems, which recognize system-specific sites inside the trapping cassette. The mechanisms of these two systems are similar, including DNA recombination in an irreversible way by strand cleavage, exchange and ligation. The targeted sites, LoxP (locus of crossover (x) in P1) and FRT (FLP-recombinase recognition target), share common structure, as they consist of two inverted repeats of 13 bp size, flanking an asymmetrical core of 8 bp (Branda and Dymecki 2004; Skarnes et al. 2011; Stanford et al. 2001; Testa et al. 2004).

The ‘Knockout-first’ allele (tm1a as named mostly), whose design is based upon the structure of gene-trap vector, takes advantage of these previous advancements and it is proposed, as its name suggests, to behave as a null allele from the beginning without any further modifications. As it is possible to convert it into a conditional allele (tm1c) by FLP-recombinase in ES cells or by breeding with transgenic FLP mice, restoring thereby its normal function as wild type allele, this system can possess the characteristics of knockout and conditional alleles in one mouse. The conditional allele after that, taking advantage of Cre-recombinase, can be reverted into true null allele (tm1d) by deleting the critical exon. This is achieved through the generation of frame-shift mutation and nonsense-mediated decay of the mutated protein, which can

22 be controlled temporarily and spatially according to the Cre mice used (Figure 1.1).

LacZ-tagged null allele (tm1b) can also be obtained directly from the ‘Knockout-first’

allele by Cre-recombinase. The trapping cassette is inserted in one of the introns of the gene of interest, which should avoid causing deletion of regulatory elements in that region, and this needs a lot of efforts to understand the structural details of the targeted gene. Computer programs are usually used applying algorithms that predict the most suitable site for the insertion of the trapping cassette and the homologous recombination strategy around the critical exon. This exon is usually chosen to be the 5’-most exon that is common to all mRNA isoforms and its deletion can cause disruption of at least 50% of the protein structure. These designing criteria are only applicable in 60 % of protein-coding genes (Skarnes et al. 2011;Testa et al. 2004).

Figure 1.1: The ‘Knockout-first’ allele structure and its possible allelic series (Adapted and modified from Ryder et al. 2013).

This allele is the most common type of mutated alleles in the collection of embryonic stem cells (ES) of the European conditional mouse mutagenesis program and the knockout mouse program (EUCOMM/KOMP-CSD), which represents the main source for generating targeted mutations in mice by the International Mouse Phenotyping Consortium (IMPC). The aims of this program are to produce knockout models for all protein-coding genes in the mouse (Brown and Moore 2012; Ryder et al. 2013; Skarnes et al. 2011), and the ‘Knockout-first’ mice were recently used in several studies as a knockout model without further changes by FLP or Cre mice (Maguire et al. 2014; Nijnik et al. 2012; Rainger et al. 2011; Wheway et al. 2013).

tm1d Null allele

tm1c Conditional allele

tm1b LacZ-tagged null allele

‘Knockout-first’

allele En2 SA IRES LacZ pA

En2 SA IRES LacZ pA hβactP neo pA

FRT site LoxP site exon c critical exon

FLPCreCre

tm1a

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