Mouse Knockouts: Modifying the Mouse Genome by Using Embryonic Stem Cells

Abstract

Mouse embryonic stem (ES) cells are derived from the early embryo. They are pluripotent and can be genetically manipulated in vitro. This allows the modification of the mouse genome by gene targeting through homologous recombination and by gene trapping approaches. Thus, mouse mutants with any desired mutation can be created and represent a valuable tool to give new insights into normal and pathological processes, providing animal models for human genetic diseases. However, until recently such a genetic manipulation was limited to the mouse. New insights into the molecular mechanisms controlling pluripotency have led to the derivation of ES cells from different species, as well as from somatic cells (iPS). In addition, the discovery of designer nucleases has opened new avenues to edit the genome and generate any knockout/knockin animal for the gene of interest. Moreover, these custom nucleases are used to introduce mutations without the requirement of ES cells.

Key Concepts

  • Embryonic stem cells represent inner cell mass (ICM) cells that are maintained at the ground state of pluripotency.
  • The repair machinery of induced double‐strand breaks is the driving mechanism allowing the design of specific mutations for the gene of interest.
  • Homologous recombination allows the design of any desired genome editing.
  • Double‐strand breaks enhance the frequency of homologous recombination events.
  • The CRISPRs/Cas9 system can be used to modify the genome of any organism and therefore its application may touch some ethical and environmental issues.

Keywords: mouse; embryonic stem cells; chimaera; knockout; homologous recombination; iPS cells; custom nucleases; ZFNs; TALENs; CRISPRs/Cas9

Figure 1. Generation of knockout mice by gene editing via pluripotent cell route or using custom nucleases. (a) The establishment of pluripotent cells can be achieved by two different routes: culture of inner cell mass (ICM) cells will give rise to embryonic stem (ES) cells, and the forced expression of Yamanaka factors in differentiated cells such as fibroblasts will generate induced pluripotent stem (iPS) cells. Initially, the Yamanaka factors were cMyc, Oct4, Sox2 and KLF4, and subsequently several modifications of the procedure were applied, including the use of chemical compounds (Shi et al., ). (b) Following genetic modification, ES and iPS cells can be used to generate chimaeric mice by blastocyst injection or morulae aggregation. The injected and aggregated embryos are transferred to foster mothers. When the born embryos are partially derived from the pluripotent ES or iPS cells, the coat colour is mixed, as schematically shown by the presence of brown colour. These mice are called chimaeras. Following mating to normal wild‐type mice they often do transmit the ES or iPS cell genome to the germ line. Custom nucleases are now used (also in conjunction with homologous recombination, see also Figure) to create genetically modified mice, such as knockout and knockin mice. This can be used either by ES cell technology or (c) via microinjection of genome‐modifying components (e.g. CRISPRs/Cas9 into the mouse zygote or as recently shown via GONAD (Takahashi et al., ). ICM, inner cell mass and TE, trophectoderm; GONAD, genome editing via oviductal nucleic acids delivery; E0.6, The morning following the detection of vaginal plug is defined as embryonic 0.5 (E.5).
Figure 2. Targeting vectors. There are two types of targeting vectors which are depicted in (a) and (b). (a) Replacement‐type targeting vector which is linearised outside of the region of homology. The vector sequences are colinear with sequences of the endogenous gene. Homologous recombination between genomic and targeting vector sequences leads to the replacement of a portion of locus of interest with targeting vector sequences. Sequences lying outside of the homologous region are excluded from the integration. Exons are shown in boxes and labelled by letters. Broken lines indicate plasmid sequences. (b) An insertion‐type vector which is linearised within the region of homology to endogenous sequences. The homologous recombination at the double‐strand break in exon 2 results in the insertion of the whole vector, including plasmid sequences (broken lines), and leading to a partial duplication of the targeted gene. Numbers indicate exons; sequences homologous to the gene to be targeted are represented by a thick line, letters indicate restriction enzyme sites. Neo is the selection marker neomycin resistance gene.
Figure 3. Conditional gene inactivation procedure. A gene targeting experiment using the Cre/LoxP and Flp/Frt recombination systems is schematically shown. Exons are indicated by closed boxes and labelled from E1 to E5. (a) Scheme displaying the genomic structure of the wild‐type allele. (b) The targeting construct, where the neomycin resistance gene is inserted in the second intron. It is flanked by the Frt sites and contains one LoxP site (black arrow); a second LoxP site is inserted into the first intron. (c) The mutated allele after homologous recombination. At this stage, chimaeric mice are generated, and the neomycin gene is removed after introducing the mutation into the mouse germ line. This is done by crossing the floxed mice to transgenic mice expressing the Flp recombinase under the control of a ubiquitous promoter (such as Rosa26Flp mice). (d) Following Flp recombination mice carrying the allele with two LoxP sites flanking exon 2 can be further crossed to transgenic mice expressing the Cre recombinase under a tissue‐specific promoter to achieve inactivation of the gene of interest in a desired tissue as shown in (e) and leading to the deletion of exon 2. Homologous regions to the locus to be targeted are indicated at the 5' and 3' end flanking the neo. In targeting construct, HSVTk is the thymidine kinase gene used for the negative selection with ganciclovir (cells which have integrated the tk gene do not survive selection with ganciclovir). neo, neomycin (cells which have integrated the neomycin resistance gene survive selection with geneticin (G418)).
Figure 4. Site‐specific recombination in mice. In the first step, a knockout mouse is generated where the gene of interest is functional but flanked by LoxP sites ‘floxed allele’ (mouse b). Before performing the specific gene inactivation, the Cre mice that will be used are crossed to the knockout mice (generated from the floxed mice by crossing to ubiquitous Cre), to get transgenic mice expressing Cre and one knockout allele (+/−), as shown for mouse (a). When a female mouse (b) is crossed to a transgenic male mouse (a), expressing a Cre transgene driven by a specific promoter (P), and carrying one knockout allele of the gene of interest, mice that are born exhibit the genotypes depicted in the animals (c), (d), (e) and (f). The desired specific inactivation occurred in mouse (c). When the Cre recombinase is used with an inducible system (hormone or tetracycline), the inactivation of the gene can be performed in a specific organ or tissue at a certain stage of development or at a postnatal stage. The same strategy could be followed using an inducible Cre that is driven by a ubiquitous promoter. fl, floxed; +/−, heterozygous for the null knockout allele.
Figure 5. Overview of genome editing using custom nucleases. The common denominator of custom nucleases is the induction of double‐strand breaks (DSB) shown in (a) where DNA is schematically represented by a ladder in blue. Region of DSB is labelled in red. (b) Following DSB, DNA repair machinery is activated, and nonhomologous end‐joining (NHEJ) mechanism results in the generation of deletions (Del) or insertions (Ins). Shown are: 1‐ and a 2‐base pair (bp) deletions, and a 1‐bp insertion. When, in addition to the custom nuclease system used, a construct with homologous domains to the target sequence is provided (shown here with an insertion of sequences coding for the green fluorescent protein), will modify the locus by inserting GFP coding sequences by homology‐directed repair (HDR). (c) Scheme displaying three designed zinc‐finger proteins (ZFPs), where each ZFP recognises 3 bp DNA sequence. In a typical experiment, 3–6 ZFPs are generated and assembled to a single ZFN by hooking it to the nuclease domain of the Fok1 restriction enzyme. As Fok1 acts as dimer, two ZFNs are necessary to build the active enzyme. The two binding monomers are separated by spacers of 5–7 bp (Kim and Kim, ). (d) Similarly, TALEs proteins are assembled and hooked to the Fok1 nuclease domain, and two TALENs are also needed to generate the active Fok1 enzyme. Shown are 12 repeat‐variable di‐residue (RVD) consisting of two amino acids each that bind to a single nucleotide. In addition, the design of TALENs requires a Thymidine at the 5′ end of the target sequence (Kim and Kim, ). (e) The CRISPRs/Cas9 nuclease system is schematically shown in light blue, with the guide RNA (gRNA) located immediately upstream of the PAM sequence at the target locus. Below the cleaved DNA strands are displayed, and the excision occurs 3 bp upstream of the PAM motif. The PAM motif is shown as TGG. While GG is necessary in the target for the induction of the nuclease activity, T can be replaced by any nucleotide.
Figure 6. Gene trap and promoter trap vectors. The principles of gene trap and promoter trap strategy are outlined in (a, b and c) and (d, e and f), respectively. In both cases, a fusion transcript is the result of the insertion mutagenesis and leads to a fusion protein of the coding sequences of the trapped gene and the reporter (c and f). This allows the expression pattern of the new identified gene to be monitored. In addition, its function is inactivated, and one can proceed for its cloning by PCR‐based techniques. The selection marker, the neomycin resistance gene, may be used with an independent promoter or can be integrated as a fusion protein to the β‐galactosidase gene (β‐gal) (not shown here). In this case only those genes are accessible to trapping that are expressed in undifferentiated ES cells. Usually strong promoters such as PGK or β‐actin are used to control neomycin expression. Px represents the coding sequences of the trapped gene.
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Mansouri, Ahmed(May 2018) Mouse Knockouts: Modifying the Mouse Genome by Using Embryonic Stem Cells. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002668.pub3]