Genetics Embryonic Stem Cells

Question: Discuss the options that are available to produce genome modifications in the?mouse using mouse embryonic stem (ES) cells (20 marks). Describe in detail a non-ES?cell method you could use to conduct a forward genetic screen in the mouse. Use diagrams to illustrate your answer. Answer: Embryonic stem cells (ESCs) are recognized as pluripotent stem cells that are derived from the inner cell mass of blastocyst in the mammalian system and have the property to differentiate into any type of cells along with additional feature of propagation and plasticity. These cells are capable of exhibiting long term proliferative potential, maintain high telomerase activity and possess a normal karyotype. Pertaining to their plasticity and potential for unlimited capacity for self renewal, ESCs are widely used in regenerative medicine and replacement of damaged tissue following injury or disease. In mouse related or murine ESCs , these characteristic features of the ESCs are utilized as well to achieve the necessary outcomes associated to genomic modifications in mouse. The genetic modifications in the context of genetic engineering generally comprise of three broad categories encompassing somatic, germline and cloning. The genome modifications generally result due to error in the DNA damage repair pathway that account for non-homologous end joining (NHEJ) and eventuallt cause double strand break (DSB) of the DNA that in turn might alter the cellular pathway to a large extent. Mouse ESCs make use of the high fidelity homologous recombinational repair (HRR) strategy to repair the DSB. Mammalian gene manipulation has been conducted by virtue of gene targeting using the murine ESCs in an effort to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene via homologous recombination (Doetschman et al. 1986). The regulation of genetic expression achieved without altering the DNA sequence is normally executed via epigenetic plasticity. Empirical research suggest that mouse ESCs express high levels of Ten-Eleven translocation (TET1) protein that mediate the induction of 5 hydroxymethyl cytosine (5hmC) upon gene regulatory elements. Further analysis showed that co-localization of 5 hmC with Polycomb repressive complex 2 (PRC 2) is very much spec ific to mouse ESCs and PRC2 dependent engagement of Tet1 offer epigenetic plasticity (Neri et al. 2013). Mouse ESCs have been demonstrated to induce pluripotent stem cells through introduction of various transcription factors as well as upregulation of certain genes. Essentially the experiment conducted by Takahashi and Yamanaka 2006, showed the potential of four factors namely Oct3/4, Sox2, c-Myc, Klf4 under adequate ES cell culture conditions induced the generation of pluripotency in somatic cells. Hence, these marker genes were identified to be crucial in genetic modifications of such cells though incorporation of certain defined factors. Expression of several other genes including Nanog, Stat3 and -catenin were found to be specific in case of the mouse ESCs as well. Further studies prove that CRISPR/Cas-9 mediated genome engineering simultaneously target five genes such as Tet 1,2, 3, Sry, Uty-8 alleles in murine ESCs via high fidelity gene editing procedures. The one step gener ation of animals carrying mutations in multiple genes by means of operating CRISPR/Cas system within the mouse ESCs are therefore found to be beneficial in studying the epistatic interactions in conjunction with in vivo analysis of the functionally redundant genes (Wang et al. 2013). Thus through the adoption of the mouse ESCs into clinical research experiments it is now possible to carry out genetic modifications ranging from subtle mutations such as point mutation, insertion or micro deletion along with chromosomal rearrangements like duplication, translocation and macro deletion through proper gene targeting methods. Fig: Genetic modifications using mouse embryonic stem cells (ESCs) (Source: www.physiolgenomics.physiology.org. (2016) Retrieved on 9 December 2016 from https://physiolgenomics.physiology.org/content/34/3/225) In the recent times, the concept of forward genetics has been rendered as a coveted topic in terms of evaluating the genetic basis responsible for expressing a specific type of phenotype in an organism. Primarily naturally occurring mutations or induction of mutation by virtue of radiation, chemicals or using transposable elements may be conducted by means of harboring this technique where the function of a gene and its phenotypic character through analysis of the altered DNA sequences may be studied. In the context of the mouse, forward genetics has been applied to expose the yet unknown genes and also in order to detect the behavioral mutant of the circadian rhythm. The advent of the high density genetic maps together with the onset of the physical mapping resources, positional cloning associated with any mutation in the mouse is a practically possible and feasible (Takahashi, Pinto and Vitaterna 1994). Relevant researches have shown the efficacy of the CRISPR-Cas 9 system as a pre cise gene targeting and gen screening tool by completely disrupting the target genes compared to other contemporary methods. Hence systematic genetic analysis is fostered by means of following this technique as evident in the mammalian cells such as that in the mouse non-embryonic stem cells (non ESCs) (Wang et al. 2014). Non embryonic stem cells or adult stem cells play a vital role in forward genetics to assess the phenotype in the diseased organism. The adult stem cells are thought to be undifferentiated cells residing in stem cell niche found among differentiated cells of the tissue organ. The maintenance and repair of the tissues in which the non-embryonic stem cells are found constitute the major functions of these cells and have the ability to give rise to some or nearly all of the bodily tissues because of their multipotent activity. A study relevant to the non-embryonic stem cells provided the evidence for differentiation of these cells into cells having a pancreatic lineag e thereby showcasing the efficacy of the forward genetics concept (Verfaillie et al. 2013). A forward genetic screen in mouse utilizing the non-ESCs method was carried out to study the sleep behavior in mice as a whole that may be referred to in this regard. At first an electroencephalogram or electromyogram based screening method of randomly mutagenised mice were done to detect the dominant mutations that influence the sleep and wakefulness. A significant reduction in the total wake time because of an increased necessity to sleep was evident following a splicing mutation in the Sik3 protein kinase gene. SIK3 was detected to have a role in the homeostatic regulation of the amount of sleep. Further probe in this matter revealed that increased excitability and discharge of the REM inhibiting neurons cause for the decline in the total amount and duration of REM sleep owing to a missense, gain of function mutation in the sodium leak channel (NALCN). Hence, the sleep pattern behavior in mice may be substantially studied and the role of SIK3 and NALCN may be comprehensively understood in regulating the NREM and REM sleep through forward genetics analysis utilizing the non embryonic murine stem cells (Funato et al. 2016). References Doetschman, T., Gregg, R.G., Maeda, N., Hooper, M.L., Melton, D.W., Thompson, S. and Smithies, O., 1986. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells.Nature,330(6148), pp.576-578. Funato, H., Miyoshi, C., Fujiyama, T., Kanda, T., Sato, M., Wang, Z., Ma, J., Nakane, S., Tomita, J., Ikkyu, A. and Kakizaki, M., 2016. Forward-genetics analysis of sleep in randomly mutagenized mice.Nature,539(7629), pp.378-383. Neri, F., Incarnato, D., Krepelova, A., Rapelli, S., Pagnani, A., Zecchina, R., Parlato, C. and Oliviero, S., 2013. Genome-wide analysis identifies a functional association of Tet1 and Polycomb repressive complex 2 in mouse embryonic stem cells.Genome biology,14(8), p.1. Takahashi, J.S., Pinto, L.H. and Vitaterna, M.H., 1994. Forward and reverse genetic approaches to behavior in the mouse.Science (New York, NY),264(5166), p.1724. Takahashi, K. and Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.cell,126(4), pp.663-676. Verfaillie, C.M., Velez, M.A.B. and Heremans, Y.P., Regents Of The University Of Minnesota, 2013.Differentiation of non-embryonic stem cells to cells having a pancreatic phenotype. U.S. Patent 8,409,859. 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. Wang, T., Wei, J.J., Sabatini, D.M. and Lander, E.S., 2014. Genetic screens in human cells using the CRISPR-Cas9 system.Science,343(6166), pp.80-84.