Disclaimer: This is an example of a student written essay.
Click here for sample essays written by our professional writers.

Any scientific information contained within this essay should not be treated as fact, this content is to be used for educational purposes only and may contain factual inaccuracies or be out of date.

Use of Knockout Mouse in CRISPR and Human Disease

Paper Type: Free Essay Subject: Biology
Wordcount: 2533 words Published: 8th Feb 2020

Reference this


A knock out mouse is a laboratory animal model in which one or more genes have been inactivated/turned off by researchers and replaced by an artificial or synthetic gene (Frese & Tuveson 2007). Gene knockout is done by deletion of gene portions in a DNA sequence. Consequently, physical and biochemical features changes in the mouse’s phenotype occur due to the loss of gene activity (Skarnes et al.2011). Knockout mice are instrumental in studying the effects of the absence of a particular gene in other animals and give information about the functioning of a knock out gene (Li et al.2013).

Knock out mouse in cancer

Knockout mice are used in understanding pertinent issues regarding the mechanisms of cell division, cell differentiation, and apoptosis. These mice are used to provide insights into the complexity of oncogenic events contributing to tumorigenesis (Skarnes et al.2011). As animal models, knockout mice are used to determine the multiple pathways leading to loss of cell cycle control and cancer pathogenesis (Carnero & Paramio 2014). Therefore, these models aid in the provision of a tractable experimental system for studying the genetics of cancer and cancer therapies.


The IEC specific hnRNPI knockout mice and severe inflammation at P14

In determining the functioning of the mammalian intestines, in this case, humans, knockout mice were used to determine the role of IECs in mammals. It was noted that the expression of the hnRNPI protein was downregulated at the IECs of knockout mice in a dramatic way. Histological analysis indicated that the entire knockout mice had moderate to severe colonic epithelium inflammation. These phenotypes are similar to the pathological characteristics of the human ulcerative colitis. In knockout mice, the colitis phenotype was noted in all colon parts whereby the distal colon was severely affected. It was also observed that young knockout mice developed neoplasmic and invasive colorectal cancer (Jin et al. 2017). Several adenomatous lesions on the epithelium of the colon in these mice exhibited dysplasia at variable degrees (Fahs et al., 2014). The p23 mice were the youngest mice to develop the adenomas.

Fig.1: Results severe inflammation at P14 was observed in the IEC-specific hnRNPI knockout mice (Jin,2017)

The HE-stained sections in figure 1 section A and B, show the severity of the inflamed colonic epithelium in P14 knockout mice in B, C, and D. The anti- Ki67 immunohistochemical staining reveals hyperproliferation at the epithelium of the knockout mice colon. Immunofluorescence staining at E and F with anti-ZOI antibody reveals an impairment of the tight junctions in the mice’s colonic epithelium. Universal eubacterial probe FISH at G and H indicates bacterial infiltration in the colonic epithelium. Counterstaining of nuclei with DAPI occurred in figure E to H. Immunohistochemical staining at I, and N shows a higher number of inflamed cells in the knockout mice lamina propria.CD4 positive cells are shown in I and J while macrophages are shown at K, and L. Neutrophils are shown at M and N.

Get Help With Your Essay

If you need assistance with writing your essay, our professional essay writing service is here to help!

Essay Writing Service

In the determination of colonic inflammation at the colonic inflammation in knockout mice, histological analysis was performed to observe the severity of inflammation at the p14. Bacterial infiltration is consistently detected, and a large number of infiltrated cells of the adaptive and innate immunity are detected at the lamina propria. The above defects are accompanied by IECs hyperproliferation (Jin et al. 2017).

How knockout mice are generated?

The vector will be designed whereby, the two markers Neo and TK are incorporated into the target gene sequence to come up with the targeting vector sequence (Fig1a) (Frese & Tuveson 2007). Step two involves the incorporation of the targeting vector into the cells of the ES. In some cells, recombination occurs between the targeting vector and the target gene knocking out a single copy of the target gene (Skarnes et al.2011). However, in other cells, recombination of the targeting vector occurs at the random chromosome section. Thirdly, cells that have successfully inserted the targeting vector into their genome are selected by treating the cells with neomycin and ganciclovir (Liu, Jenkins and Copeland 2003). The only cells that survive after this treatment are the ones that have incorporated the targeting vector into the target gene. The fourth step is the injection of the selected cells into a normal and developing mouse embryo resulting in a chimera (Li et al.2013). The chimeric mouse containing a mixture of its cells and the heterozygous knock out cells are bred with normal mice. The knock out gene is then passed into the resulting offspring (Fig1b).

The CRISPR technology is one of the many laboratory research techniques that are revolutionizing clinical and biological research (Wang et al.2013). The technology helps create animal and cell models that are used in the validation of identified variants, pathogeny characterization, and determination of the functionality of genes. It also provides solutions for correcting molecular defects. The CRISPR technique has been used to create knockout mice. The technique involves model design and production of the CRISPR/Cas9 nuclease which considers the target features of a gene (Wang et al.2013). The technique also involves injecting CRISPR/Cas9 nuclease into fertilized mouse embryos and re-implanting these embryos into a surrogate mother to produce founder mice.




Fig2: Diagram a: Image displaying the process of producing target vector using the markers. Diagram b displays the process of creating the knockout mice after introducing target vector to ES cells.


The CRISPR procedure for the generation of knockout mice

The first step is the design of a nuclease-mediated strategy which includes selecting the target sites in a gene based on an optimized algorithm (Wang et al.2013). The optimized algorithm does maximize the activity on the target nuclease, minimize off-target activity and design nuclease expression vectors. In ensuring that the procedure is successful, a vector is designed against two target sites for every gene that is to be knocked out or deleted (Sung et al.2014). Therefore, the DNA vectors constructed that express the desired nuclease will be tested for efficacy in cell culture. The second step is injecting nuclease into the mouse eggs whereby the transcription of the nuclease expression vectors is first done in vitro, and the resulting mRNA is artificially capped to facilitate translation (Liu et al. 2003). The fertilized eggs injected with the nuclease are implanted into a surrogate mother which results into the generation of founders. Founder screening techniques like PCR and sequencing are then used to identify knockout founder mice (Hamilton et al. 2014). Mice with frameshift deletions and insertions are considered knock out founders. The founder mice are bred then characterized and genotyped. Assessment is then carried out to detect off-target mutations (Khor et al.2008).  The knockout mice produced by the CRISPR technique can be used to investigate male infertility regarding mutations on the sperm flagella morphology. Hence the CRISPR technique is a timely and efficient strategy for identifying and validating mutations causing male infertility in humans.

Gene trapping and gene targeting and other knock out methods

Gene trapping is a method used in the creation of knockout mice by making use of Embryonic Stem cells (Sung et al.2014). In this method, a random process of introducing an artificial gene is involved. Consequently, the insertion event leads to a knock out of the native gene. A synthetic reporter gene designed to impair the natural splicing process is inserted into the DNA of a cell randomly (Lader 2015). Through the reporter gene, researchers can get information about the expression and function of the interrupted mouse genes (Wang et al.2013). Subsequently, early-stage mouse embryos are then injected with the gene trap modified ES cells which leads into the production of gene trap knockout mice.

Find Out How UKEssays.com Can Help You!

Our academic experts are ready and waiting to assist with any writing project you may have. From simple essay plans, through to full dissertations, you can guarantee we have a service perfectly matched to your needs.

View our services

Conversely; gene targeting is dependent on methods used for inducing mutations in mouse genes (Sung et al.2014). The process involves homologous recombination where an artificial gene sequence is introduced based on target gene sequences. Cellular homologous recombination takes over in the Embryonic Stem cells and identifies equal parts in a series (Kazdoba et al. 2014). The original piece of DNA is then replaced with the engineered knock out gene sequence creating knockout mice. Zinc finger nucleases are proteins of a chimeric nature consisting of an endonuclease domain fused to a binding domain (Carbery et al, 2010).

Zinc finger nucleases cause dimerization which causes double-strand breaks in a target DNA. They are therefore a single step approach used to generate knockout mice by performing non-homologous end joining, after cleavage of the target DNA (Carbery et al, 2010). Non-homologous end joining is, however, an error-prone process of DNA repair which introduces insertions and deletions.

Comparison of gene targeting, gene trapping, CRISPR and Zinc Finger Nucleases

CRISPR is an easy to use, efficient and straightforward method of creating knockout mice as compared to other methods. It also reduces the time required for gene modifications (Carnero & Paramio 2014). Compared to other methods; CRISPR has the highest success rate for the incorporation of the desired mutation. Therefore, CRISPR has considered the best gene knock out method hence its popularity in scientific research (Liu et al.2003). Gene finger nuclease has high efficiency and eliminates the risk of integration of random genome. Compared to other methods it removes the need for nucleases and nuclear delivery (Austin et al. 2004).

In Conclusion, CRISPR gene editing models have consistently transformed transgenesis technology.  It possible that all conventional step to effectively develop knockout alleles in mice will be circumvented in the future. The CRISPR will revolutionize mice knockout process through delivery of reagents in situ into oviducts.


  • Austin, C.P., Battey, J.F., Bradley, A., Bucan, M., Capecchi, M., Collins, F.S., Dove, W.F., Duyk, G., Dymecki, S., Eppig, J.T. and Grieder, F.B., 2004. The knockout mouse project. Nature genetics36(9), p.921.Retrieved from: https://www.nature.com/scitable/content/the-steps-involved-in-making-a-knockout-7334330
  • Carnero, A. and Paramio, J.M., 2014. The PTEN/PI3K/AKT pathway in vivo, cancer mouse models. Frontiers in oncology4, p.252.
  • Fahs, S.A., Hille, M.T., Shi, Q., Weiler, H. and Montgomery, R.R., 2014. A conditional knockout mouse model reveals endothelial cells as the predominant and possibly exclusive source of plasma factor VIII. Blood, pp.blood-2014.
  • Frese, K.K. and Tuveson, D.A., 2007. Maximizing mouse cancer models. Nature Reviews Cancer7(9), p.654.
  • Hamilton, A., Esseltine, J.L., DeVries, R.A., Cregan, S.P. and Ferguson, S.S., 2014. Metabotropic glutamate receptor 5 knockout reduces cognitive impairment and pathogenesis in a mouse model of Alzheimer’s disease. Molecular brain, 7(1), p.40.
  • Kazdoba, T.M., Leach, P.T., Silverman, J.L. and Crawley, J.N., 2014. Modeling fragile X syndrome in the Fmr1 knockout mouse. Intractable & rare diseases research, 3(4), pp.118-133.
  • Khor, T.O., Huang, M.T., Prawan, A., Liu, Y., Hao, X., Yu, S., Cheung, W.K.L., Chan, J.Y., Reddy, B.S., Yang, C.S. and Kong, A.N., 2008. Increased susceptibility of Nrf2 knockout mice to colitis-associated colorectal cancer. Cancer prevention research, pp.1940-6207.Retrieved from: t https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954478/
  • Lader, M., 2015. Generalized anxiety disorder. In Encyclopedia of psychopharmacology (pp. 699-702). Springer, Berlin, Heidelberg.
  • Li, D., Qiu, Z., Shao, Y., Chen, Y., Guan, Y., Liu, M., Li, Y., Gao, N., Wang, L., Lu, X. and Zhao, Y., 2013. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nature biotechnology31(8), p.681.Retrieved from:http://www.genome-engineering.org/crispr/
  • Liu, P., Jenkins, N.A. and Copeland, N.G., 2003. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome research13(3), pp.476-484.Retrieved from:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371387/
  • Skarnes, W.C., Rosen, B., West, A.P., Koutsourakis, M., Bushell, W., Iyer, V., Mujica, A.O., Thomas, M., Harrow, J., Cox, T. and Jackson, D., 2011. A conditional knockout resource for the genome-wide study of mouse gene function. Nature474(7351), p.337.
  • Sung, Y.H., Kim, J.M., Kim, H.T., Lee, J., Jeon, J., Jin, Y., Choi, J.H., Ban, Y.H., Ha, S.J., Kim, C.H. and Lee, H.W., 2014. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome research24(1), pp.125-131.
  • Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. and Gregory, P.D., 2010. Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics11(9), p.636.
  • Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F. and Jaenisch, R., 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. cell153(4), pp.910-918.


Cite This Work

To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Related Services

View all

DMCA / Removal Request

If you are the original writer of this essay and no longer wish to have your work published on UKEssays.com then please: