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KNOCKOUT MOUSE AND HUMAN DISEASE

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Introduction

A knock out mouse is a laboratory animal model in which one or more genes have been

inactivated or 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 have been instrumental in understanding pertinent issues regarding the

mechanisms of cell division, cell differentiation, and apoptosis. These mice have been used to

provide insights into the complexity of oncogenic events contributing to tumorigenesis (Skarnes

et al.2011). As animal models, knockout mice have been instrumental in determining the

multiple pathways leading to loss of cell cycle control and cancer pathogenesis (Carnero &

Paramio 2014). Therefore, these models are useful 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 in the determination of 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 (Urnov et al. 2010). Histological analysis indicated that the entire knockout mice

had moderate to severe colonic epithelium inflammation. These phenotypes are similar to the
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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 (Urnov et

al. 2010). It was also observed that young knockout mice developed neoplasmic and invasive

colorectal cancer. The 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.

Austin et al. (2004)

The HE-stained sections in figure 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(Austin et al. 2004).

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(Austin et al. 2004). Counterstaining of

nuclei with DAPI occurred in figure E to H. Immunohistochemical staining at I, and N shows a

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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.

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

(Austin et al. 2004). 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.

How knockout mice are generated?

The first step is the designing of the targeting vector whereby, the two markers Neo and

TK are incorporated into the target gene sequence to come up with the targeting vector sequence

(Frese & Tuveson 2007). The second step 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.

The CRISPR technology is one of the many laboratory research techniques that are

revolutionizing clinical and biological research (Wang et al.2013). The technology allows for the
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creation of 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 for the creation

of knockout mice. The technique involves model design and production of the CRISPR/Cas9

nuclease which takes into account the target features of a gene (Wang et al.2013). The technique

also involves the injection of the CRISPR/Cas9 nuclease into fertilized mouse embryos and

reimplantation of these embryos into a surrogate mother to produce founder mice.

The diagrams below give an overview of the entire process. The five steps are depicted

below and so have the diagrams drawn through Inkscape.

Drawn from: Inkscape

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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 the; maximization of the activity on the target nuclease, minimization of off-

target activity and design of 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 the injection of the 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.
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Sourced from Khor et al. (2008)

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.

Conversely; gene targeting is dependent on methods used for inducing mutations in

mouse genes (Sung et al.2014). The process involves homologous recombination whereby an
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artificial gene sequence is introduced based on target gene sequences. Afterward, 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 (Khor et

al.2008). 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 (Liu, Jenkins and Copeland

2003). Non-homologous end joining is, however, an error-prone process of DNA repair which

introduces insertions and deletions. The diagram below shows the mechanism of action of zinc

finger nucleases.

Sourced from Liu, Jenkins and Copeland (2003)

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

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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). Additionally, 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).

Conversely, 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).

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References

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 genetics, 36(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 oncology, 4, 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

Cancer, 7(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/

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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 biotechnology, 31(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 research, 13(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. Nature, 474(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 research, 24(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 Genetics, 11(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. cell, 153(4), pp.910-918.