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


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.


Blair K, Wray J and Smith A (2011) The liberation of embryonic stem cells. PLoS Genetics 7 (4): e1002019. DOI: 10.1371/journal.pgen.1002019.

Boettcher M and McManus MT (2015) Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Molecular Cell 58 (4): 575–585.

Buehr M, Meek S, Blair K, et al., (2008) Capture of authentic embryonic stem cells from rat blastocysts. Cell 135 (7): 1287–1298.

Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty‐first century. Nature Reviews Genetics 6: 507–512.

Carbery ID, Ji D, Harrington A, et al., (2010) Targeted genome modification in mice using zinc‐finger nucleases. Genetics 186 (2): 451–459.

Cermak T, Doyle EL, Christian M, et al., (2011) Efficient design and assembly of custom TALEN and other TAL effector‐based constructs for DNA targeting. Nucleic Acids Research 39 (12): e82. DOI: 10.1093/nar/gkr218.

Cong L, Ran FA, Cox D, et al., (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823.

Gaj T, Gersbach CA and Barbas CF et al. (2013) ZFN, TALEN, and CRISPR/Cas‐based methods for genome engineering. Trends in Biotechnology 31 (7): 397–405.

Gurumurthy CB, Takahashi G, Wada K, et al., (2016) GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos. Curr Protoc Hum Genet. 88: Unit 15.8. DOI: 10.1002/0471142905.hg1508s88.

Harrison MM, Jenkins BV, O'Connor-Giles KM, et al., (2014) A CRISPR view of development. Genes & Development 28 (17): 1859–1872.

Horvath P and Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327 (5962): 167–170.

Jaenisch R (1988) Transgenic animals. Science 240 (4858): 1468–1474.

Jinek M, Chylinski K, Fonfara I, et al., (2012) A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821.

Jinek M, East A, Cheng A, et al., (2013) RNA‐programmed genome editing in human cells. eLife 2: e00471. DOI: 10.7554/eLife.00471.

Kim H and Kim JS (2014) A guide to genome engineering with programmable nucleases. Nature Reviews Genetics 15 (5): 321–334.

Lin Y, Cradick TJ, Brown MT, et al., (2014) CRISPR/Cas9 systems have off‐target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42 (11): 7473–7485.

Mali P, Yang L, Esvelt KM, et al., (2013) RNA‐guided human genome engineering via Cas9. Science 339 (6121): 823–826.

Marraffini LA and Sontheimer EJ (2010) CRISPR interference: RNA‐directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 11 (3): 181–190.

Pattanayak V, Guilinger JP and Liu DR (2014) Determining the specificities of TALENs, Cas9, and other genome‐editing enzymes. In: Doudna JA and Sontheimer EJ (eds) Methods in Enzymology, vol. 546, pp. 47–78. Academic Press, Elsevier: San Diego, CA.

Petersen B and Niemann H (2015) Molecular scissors and their application in genetically modified farm animals. Transgenic Research 24: 381–396.

Rouet P, Smih F and Jasin M. (1994) Expression of a site‐specific endonuclease stimulates homologous recombination in mammalian cells. Proceedings of National Academy of Sciences of the United States of America 91 (13): 6064–6068.

Sato M, Ohtsuka M, Watanabe S, et al., (2016) Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old‐new. Biology Direct 11: 16. DOI: 10.1186/s13062-016-0115-8.

Shi Y, Inoue H, Wu JC, et al., (2017) Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 16 (2): 115–130.

Takahashi K and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 (4): 663–676.

Takahashi K, Tanabe K, Ohnuki M, et al., (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131 (5): 861–872.

Takahashi G, Gurumurthy CB, Wada K, et al., (2015) GONAD: Genome‐editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice. Scientific Reports 5: 11406. DOI: 10.1038/srep11406.

Wang H, Yang H, Shivalila CS, et al., (2013) One‐step generation of mice carrying mutations in multiple genes by CRISPR/Cas‐mediated genome engineering. Cell 153 (4): 910–918.

Wiedenheft B, Sternberg SH and Doudna JA (2012) RNA‐guided genetic silencing systems in bacteria and archaea. Nature 482 (7385): 331–338.

Wiles MV, Qin W, Cheng AW, et al. (2015) CRISPR‐Cas9‐mediated genome editing and guide RNA design. Mammalian Genome 26: 501–510.

Further Reading

Bogdanove AJ and Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333: 1843–1846.

Capecchi MR (1989) Altering the mouse genome by homologous recombination. Science 244: 1288–1292.

Copeland NG, Jenkins NA and Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nature Reviews Genetics 2: 769–779.

Doudna JA and Sontheimer EJ (eds) (2014) Methods in Enzymology, vol. 546, pp. 47–78. San Diego, CA: Academic Press, Elsevier.

Hasty P, Abuin A and Bradley A (2000) In Gene Targeting: A Practical Approach, 2nd edn, pp. 3–35. New York: Oxford University Press.

Kuhn R and Torres RM (2001) Cre/LoxP recombination system and gene targeting. Methods in Molecular Biology 180: 175–204.

Kunath T, Gish G, Lickert H, et al. (2003) Transgenic RNA interference in ES cell‐derived embryos recapitulates a genetic null phenotype. Nature Biotechnology 21: 559–561.

Metzger D and Chambon P (2001) Site‐ and time‐specific gene targeting in the mouse. Methods. Embryonic stem cells, Methods and Protocols (2002). In: Turksen K (ed) Methods in Molecular Biology, vol. 185, pp. 71–80. Totowa, NJ: Humana Press.

Robertson EJ (ed) (1987) Embryo‐Derived Stem Cell Lines. Teratocarcinomas and Embryonic Stem Cells. A Practical Approach, pp. 71–112. Oxford, UK: IRL Press.

Sadlack B, Merz H, Schorle H, et al. (1993) Ulcerative colitis‐like disease in mice with a disrupted interleukin‐2 gene. Cell 75: 253–261.

Sauer B (1993) Manipulation of transgenes by site‐specific recombination: use of Cre recombinase. Methods in Enzymology 225: 890–900.

Stanford WL, Cohn JB and Cordes SP (2001) Gene‐trap mutagenesis: past, present and beyond. Nature Reviews Genetics 2: 756–768.

Torres RM and Kühn R (eds) (1997) Laboratory Protocols for Conditional Targeting, p. 167. Oxford, UK: Oxford University Press.

Williams B and Jacks T (1996) Mechanisms of carcinogenesis and the mutant mouse. Current Opinion in Genetics and Development 6: 65–70.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Mansouri, Ahmed(May 2018) Mouse Knockouts: Modifying the Mouse Genome by Using Embryonic Stem Cells. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0002668.pub3]