Knockout and Knock‐In Animals

Ahmed Mansouri, Max‐Planck Institute for Biophysical Chemistry, Göttingen, Germany

Published online: June 2018

DOI: 10.1002/9780470015902.a0000991.pub3

Abstract

The inactivation of a gene (knockout) or the insertion of a coding sequence into a gene of interest (knock‐in) provides genetically modified animals for the analysis of genetic circuits in developmental biology and for use as models for human disease. Recently developed approaches have facilitated the generation of mutant animals. Thus, designed nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPRs‐associated nucleases (CRISPR/Cas9), offer a platform to create knockout and knock‐in animals of various species without going through the route of embryonic stem cell technology. Custom nucleases allow the site‐specific recognition and deoxyribonucleic acid (DNA) cleavage to induce DNA repair machinery resulting in insertions and/or deletions (Indel) in the desired locus. Providing a construct with homologous sequences to the 5′ and 3′ end of the nuclease cleavage site will result in homologous recombination‐directed repair (HDR) of the gene of interest and giving rise to knock‐in animals.

Key Concepts

  • Animal behaviourists must participate in conservation planning to protect the future of biodiversity.
  • Lipid bilayers provide the fundamental architecture of biological membranes.
  • Knockout and knock‐in animals are absolutely required for the analysis of gene function.
  • Custom nucleases allow the generation of knock‐in animals from various species.
  • iPS cells may facilitate the establishment of pluripotent stem cells from various species.
  • Designed nucleases‐based gene editing provides a simple tool to create animal models that recapitulate human disease pathologies.

Keywords: transgenic mouse; embryonic stem cells; knockout; knock‐in animals; homologous recombination

Figure 1. Knock‐in targeting constructs. To illustrate the knock‐in strategy, the genomic organisation of a mammalian gene with four exons is shown (a); (b, c) Different steps in order to perform a knock‐in of a marker such as GFP or of any other cDNA of interest into the locus (a). The first exon containing the ATG is deleted and replaced by GFP coding sequences and the selection marker neo (b). In a first electroporation experiment, the targeting construct (b) is introduced into wild‐type ES cells. Homologous recombination events generate ES clones that carry a knock‐in allele, as shown in (b). Mice carrying construct in (b) are crossed to transgenic mice expressing Cre recombinase under a ubiquitous promoter to delete neo. GFP is then expressed under regulatory sequences of the locus (a). (d) A targeting construct for the generation of a floxed allele by using the Frt sites to delete the selection marker, which is flanked by two Frt sites (in parallel orientation) and carries in addition one LoxP site (arrowhead). The second LoxP site required for the floxed allele is inserted in front of the first exon. The Frt sites allow the excision of the neo gene by crossing mice carrying the mutated allele in (d) to transgenic mice that express the Flp recombinase (Dymecki, ). The Flp recombinase is expressed under a ubiquitous promoter such as Rosa26. After deletion of the neo, the LoxP and one Frt site remain in the locus (e). (e) The genomic organisation of a floxed allele generated from the gene (a), where LoxP sites are flanking the first exon. Transgenic mice expressing Cre recombinase driven by specific promoters may be then used to inactivate gene (a) (using the floxed allele) in specific tissues. External 5′ and 3′ probes for the screening of homologous recombination events are indicated in (a). (f) Scheme representing the custom nuclease CRISPR/Cas9 complex (blue) and the induction of double‐strand break. (g) Following double‐strand break, DNA repair machinery is induced: two possible outcomes are shown in (h). Nonhomologous end joining (NHEJ) will result in Indels (insertions and deletions), homologous recombination‐directed repair (HDR) gives rise to knock‐in of the offered sequence (shown as GFP with homologous arms to the 5′ and 3′ end of the locus) via homologous recombination (g,h). neo, neomycin; GFP, green fluorescent protein; PA, polyadenylation signal; Pr, promoter and bent arrow indicates transcriptional direction.
Figure 2. Generation of knockout and knock‐in animals. Scheme displaying the different routes allowing the generation of knockout and knock‐in animals. (a) Pluripotent embryonic stem (ES) cells, as well as induced pluripotent stem (iPS) cells can be used to create such animals via homologous recombination‐mediated genetic modification. Mutant cells are injected into the blastocyst to give rise to chimaeric animals that are able to transmit the modified ES/iPS genome to the germ line, when crossed to wild‐type animals. Six weeks following transfection of ES/iPS cells (a), homologous recombinant ES/iPS clones are established. Three weeks after blastocyst injection chimaeric mice are born and will be mated at 6–7 weeks of age to achieve germ line transmission. The whole process required to get heterozygous mutant animals requires about 5–6 months. (b) Custom nucleases (ZFNs, TALENs and CRISPR/Cas9 nuclease) have the potential to derive such animals with, as well as without, ES cell technology. Specifically, CRISPR/Cas9 is becoming the mostly used technique to reach this goal. Modification of the genome can be achieved by injection of gene‐editing components in the early one‐cell embryo. Three weeks following microinjection, mice are born, and genetically modified mice are identified 3 weeks later. When mutant mice reach the age of 6–7 weeks, they are ready for mating to generate homozygous animals. Of note is the alternative application of CRISPR/Cas9 nuclease gene‐editing procedure in ES cells. HR, homologous recombination.
Figure 3. Insertion of the green fluorescent protein (GFP) in the Lmx1a locus. Side view of whole mouse embryo at embryonic day 10.5 of gestation (E10.5) and heterozygous for GFP knock‐in, revealing GFP expression. GFP signal recapitulates the expression of Lmx1a in the early mouse embryo: in the roof plate, in the otic vesicle, in the tail bud and in the ventral midbrain where it is known to label dopaminergic neuron progenitors (Andersson et al., ) (see white arrow). Lmx1a is a member of LIM‐homeodomain family of transcription factors (Millonig et al., ). OV, otic vesicle; SC, spinal cord; tb, tail bud.
Figure 4. (a) Scheme representing a genetic locus where two mutated (heterospecific) LoxP sites were introduced (e.g. in ES cells) to flank the first exon which is planned to be the target of replacement. (b) A plasmid carrying green fluorescent protein coding sequences, also flanked by identical heterospecific LoxP sites (GFP), is electroporated together with a Cre expressing plasmid in the ES cells carrying the construct in (a). Following the electroporation, Cre will lead to recombination yielding the exchange of Exon 1 by GFP coding sequences. Such recombination‐mediated cassette exchange (RMCE) procedure is of interest in many experiments, allowing the easy insertion of any sequence of interest in the locus (a), as often, as required for the research project. Lpx, LoxP site. Following Cre‐mediated recombination two products are formed: (c) the gene of interest where GFP has been inserted through the recombinase‐mediated cassette‐exchange (RMCE) system and (d) where the GFP‐donor plasmid is now carrying exon one of the gene shown in a) (blue box). The donor plasmid will not integrate and will be diluted out during further cell division.
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Related Sub-Topics:

Chromosomes and Chromatin Modifications | Techniques and Tools in Molecular Biology | Model Organisms and Human Disease | Techniques and Tools in Clinical Genetics | Genetic Models
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Article Title: Knockout and Knock‐In Animals
 
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Mansouri, Ahmed(Jun 2018) Knockout and Knock‐In Animals. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000991.pub3]