Agric. Fibroblasts with modified MSNT were used as nuclear donor for SCNT. 27, 525–537. (2012). These include electroporation-, liker-, retroviral-, liposome (lipofection)-based SMGT, restriction enzyme–mediated integration, and further techniques (Smith, 2012). Cloning of the Xuhuai goat PPARγ gene and the preparation of transgenic sheep. Dev. “Handmade somatic cell cloning in cattle,” in Nuclear Transfer Protocols (Totowa, NJ: Springer), 183–195. Thrombolysis 28, 449–457. Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat. Expression of human blood clotting factor VIII (FVIII) constructs in the mammary gland of transgenic mice and sheep. doi: 10.1093/nar/30.11.2299, Archer, J. S., Kennan, W. S., Gould, M. N., Bremel, R. D. (1994). Animals are now produced to make healthier meat and more efficient milk. Transgenic animals are those that have been genetically modified. Theriogenology 78, 583–592. Cell. Handmade cloning (HMC) is a simplified version of the SCNT technique. Nature 277, 298–300. EDAR-knockout founders showed abnormal primary hair follicles and an absence of hair on the top of their heads; these characteristics are distinctive features of EDAR mutants. Humans. Chin. doi: 10.1016/j.anireprosci.2005.12.001, Vajta, G., Lewis, I. M., Hyttel, P., Thouas, G. A., Trounson, A. O. Transgenic organisms are genetically engineered to have a transgene, a new gene, often from another species, inserted into their genome so that it can produce a desired protein. During that time, in 1985, the first report about the generation of transgenic farm animals (including sheep) via pronuclear injection (PNI) was published, announcing the first procedure for the production of transgenic farm animals (Hammer et al., 1985). A posteriori FISH showed that a high proportion of nuclei from cell line A scored negatively as compared to the B–D isolates. Cloning Transgenes. *Correspondence: Xiaolong Wang, [email protected]; Yulin Chen, [email protected], Front. Nature 307, 637–638. J. doi: 10.1002/bit.24890, Sun, R., Li, Q., Yan, H., Zhao, J., Gao, X. Each chapter is written by a leader in the field. The book includes timelines and examples for each expression system, and provides an overview of the future of recombinant protein expression. (1989). Anim. This book can be used as both a reference book and a textbook for specialized university courses and will be of interest to everyone involved in basic research in animal biology, molecular genetics, animal biotechnology, pharmaceutical ... (1991). Articles, College of Sciences, Nanjing Agricultural University, China, Washington State University, United States. doi: 10.1371/journal.pone.0129457, Olorunniji, F. J., Rosser, S. J., Stark, W. M. (2016). 30, 1175–1182. Examples of CRISPR-generated Transgenic Animals. This has allowed the determination of the sequences of probably all variable (V), and certainly all diversity (D), joining (J), and constant (C) region genes (Brüggemann et al., 2015). Small Rumin. CRISPR/Cas9-mediated sheep MSTN gene knockout and promote sSMSCs differentiation. doi: 10.1111/j.1439-0388.1996.tb00634.x, Niu, Y., Jin, M., Li, Y., Li, P., Zhou, J., Wang, X., et al. A., Shah, R. K., Joshi, C. G. (2014). Humans. Further advances in this field are required to enable the widespread application of these techniques to facilitate multiplication, transgenesis, and genetic rescue of threatened genomes. doi: 10.1016/j.smallrumres.2006.12.009, Wang, X., Cai, B., Zhou, J., Zhu, H., Niu, Y., Ma, B., et al. Biotechnol. doi: 10.1016/j.molcel.2015.04.028, Bogdanove, A. J., Voytas, D. F. (2011). Commun. 92, 211–230. 23, 33–40. doi: 10.1172/jci.insight.123529, Fan, Z., Regouski, M., Yang, M., Gash, K., Meng, Q., Davies, C. J., et al. doi: 10.1016/j.jbiotec.2005.10.019, Tripathi, A. K., Ramani, U. V., Patel, A. K., Rank, D. N., Joshi, C. G. (2013). For this, ZFNs have been designed to introduce biallelic disruption in forkhead box L2 (FOXL2) to investigate the function of this gene in female sex determination (Boulanger et al., 2014). 1 year later, the same group reported pioneering and unprecedented work of utilizing fetal and adult mammalian cells to produce viable offspring, which resulted in the generation of the first and most famous somatic cloned animal in the world, the sheep Dolly (Wilmut et al., 1997). Transgenic Res. Genet. A novel recombinant human plasminogen activator: efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats. (2014). (2013). 76, 933–937. (2014a). doi: 10.1096/fj.07-8935com, Hosseini, S. M., Hajian, M., Forouzanfar, M., Moulavi, F., Abedi, P., Asgari, V., et al. Biotechnol. Chang Gung Med. Examples of generated transgenic sheep and goats using SCNT are listed in Table 2. Theriogenology 21, 555–560. doi: 10.1016/j.gene.2008.04.020, Yang, H., Li, Q., Han, Z., Hu, J., Li, W., Liu, Z. Thus a large number of functional human Ig expression gene loci are transmitted to a transgenic mouse. Fertil. Biosci. (2014). Bacterial Profiling of Raw Milk from Lysozyme Transgenic and Control Goats . (2012b). The CRISPR/Cas9 system has been applied in sheep and goats to achieve aims related to improving animal health and welfare. Gene editing has been revolutionized as a result of the rapid emergence of novel varieties of tools that can simply, precisely, and more efficiently mediate different forms of DNA modifications than previously reported tools. Other forms of modifications, such as large deletions and inversions, can also be achieved using optimized endonucleases. Tucker, E. M., Moor, R. M., Rowson, L. E. A. An open clinical trial has suggested some clinical benefits from this approach and a double‐blind study is underway. Experiments in transgenic Drosophila (Chung et al., 1993), in K562 cells (Walters et al., 1999), in adeno-associated virus (AAV) or retrovirus infected MEL cells and bone marrows (Inoue et al., 1999; Emery et al., 2000; Rivella et al., 2000), as well as in transgenic mice (Taboit-Dameron et al., 1999; Potts et al., 2000) have shown that flanking a transgene with the chicken beta-globin 5′CHS4 can significantly reduce silencing due to chromosomal position effects and promoter interference. Unfortunately these high levels of expression were incompatible with normal lactations. Found inside – Page 266Activities in goat therapeutic proteins are in progress, for example, a human factor IX is already produced in Iran but ... of transgenic goats is underway, no data on transgenic goats producing monoclonal antibodies has been published, ... The company has successfully generated distinct lines of goats that produce in their milk recombinant versions of either the MaSpI or MaSpII . (2018). Initially, CRISPR/Cas9 has been applied to target the NUP155 gene in goat fibroblasts to generate NUP155-knockout donor cells, which can be used in the SCNT program to produce NUP155-knockout goat model (Hu et al., 2014; Ni et al., 2014; Fan et al., 2019). TALENs have been applied to sheep and goats and live founders with desired genetic alterations have been generated. Biol. Biotechnol. In addition to utilizing this approach as a valuable tool in embryological studies and to achieve the multiplication of desired genetics, nuclear transfer has become one of the basic methods to generate genetically modified animals with useful and desired traits. Tongue epithelium cells from shRNA mediated transgenic goat show high resistance to foot and mouth disease virus. Menchaca et al. doi: 10.1073/pnas.91.24.11303, Brinster, R. L., Zimmermann, J. W. (1994). Generation of a transgenic cashmere goat using the piggyBac transposition system. Genet. Moreover, further studies are ongoing for the provision of useful sheep and goat models for agriculture and biomedicine. Goats are an ideal model for the production of pharmaceutical molecules in milk and have been subjected to this protocol. Rep. 6, 33675. doi: 10.1038/srep33675, Li, T., Ma, A. T., Zhang, Y. J., Liu, Y. Q., Wang, S. Y., Shi, X. F., et al. In June 2006, the first therapeutic protein made in a transgenic animal was approved for use in Europe and the USA. Sci. 24, 811–820. Biol. Because the number of blastodermal cells at stage X is as high as 60,000, the more the viral stock is concentrated, the higher the expected transgenesis efficiency. Curr. In agriculture, transgenic animals are engineered to achive better genetic characteristics such as increased production of dairy proteins for food, enhanced production of wool or enhanced disease resistance. (2004). 91, 11298–11302. Quantitative evaluation of myostatin gene in stably transfected caprine fibroblast cells by anti-myostatin shRNA. Some drugs are also made in transgenic plants, such as tobacco. Reprod. 57, 188–196. Cell 91, 501–510. Biotechnol. doi: 10.1089/clo.2005.7.178, Rodriguez-Sosa, J. R., Silvertown, J. D., Foster, R. A., Medin, J. Lysostaphin. Like Herman, each calf carries a gene for production in cow milk of human lactoferrin (HLF). It became apparent almost immediately that the method used to produce the transgenic livestock had substantial limitations that would impede its use both for research and commercial applications. For example, you can study your gene of interest by generating a transgenic mouse model with its gene expression. Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells. Biol. This ratio might be increased by improving SCNT conditions. Int. (A) The four major classes of endonucleases: meganucleases, ZFNs, TALENs, and CRISPR/Cas9. High expression level of recombinant human erythropoietin in the milk of non-transgenic goats. The most critical problem in early transgenic animal production was low infectivity of the conventional amphotropic or xenotropic MLV-based retrovirus vector viruses, especially to the cells derived from ungulates. ZFNs have been designed to mediate disruption in the goat BLG gene. This is evidenced by line A producing more than 40 g of rhA per liter of milk. Sheep cloned by nuclear transfer from a cultured cell line. L ast week while at the NISE Net network-wide meeting, I was fortunate enough to see a preview of part of NOVA's forthcoming series Making Stuff. 77, 7380–7384. Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation. The opportunities suggested in this book offer new approaches that can synergize with each other and with many other activities to transform agriculture in sub-Saharan Africa and South Asia. The use of endonucleases has attracted significant attention among scientists after the role of double-strand breaks (DSBs) in increasing the efficiency of HR event has been highlighted (Rouet et al., 1994; Choulika et al., 1995). This study was supported by the National Natural Science Foundation of China (31772571, 31572369, 31872332), and Local Grants (NXTS2018-001, 2017NY-072, and 2018KJXX-009). Animal Biology 2009. This basic research is only the beginning; the potent potential of genome engineering will become apparent in the future. In sheep and goats, initial publications have reported the application of this technique for successful embryo development (Peura and Vajta, 2003; Akshey et al., 2008), followed by a number of publications reporting the application of this technique to produce cloned (Malik et al., 2014; Khan et al., 2018), transgenically cloned (Lagutina et al., 2007; Pereira et al., 2013), or interspecies cloned embryos (Selokar et al., 2011; Yu et al., 2011). What is produced in the milk of transgenic goats? Of these gene manipulation tools, the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) is currently revolutionizing the field of genome editing throughout virtually all biological kingdoms (Doudna and Charpentier, 2014; Hsu et al., 2014). Biotechnol. doi: 10.1016/j.theriogenology.2019.03.029, Zhang, P., Liu, P., Dou, H., Chen, L., Chen, L., Lin, L., et al. The world's first cloned transgenic goats have been born as part of a research program conducted by LSU Agricultural Center and Genzyme Transgenic Corp. The resultant offspring of this process resembles nucleic donors. Reprod. Anim. Rep. 41, 1003–1013. BMC Vet. (1997). (1998) reported that injection of 10 pL of highly concentrated viral stock (109 CFU/mL) into the perivitelline space of the metaphase II bovine oocyte resulted in 100% efficiency of transgenic bovine production. Proc. Species transgenic animals examples. These genes are directly or indirectly related to milk traits and affect the fatty acid metabolism. Dev. The results of these promising attempts highlight the potential of gene-edited sheep to be applied in interspecies organ generation as candidate hosts for the growth of human organs. (2007). (1998). The fiber artificially created from this silk protein has several potentially valuable uses, such as making lightweight, strong, yet supple bulletproof vests. Abbasi, H., Hosseini, S. M., Hajian, M., Nasiri, Z., Bahadorani, M., Tahmoorespur, M., et al. 7, 171–192. Preparation of highly concentrated viral stock is the most critical factor in transgenic avian studies as well. Campbell et al. doi: 10.1007/s11248-016-9988-7, Singh, N. K., Singh, S., Jain, S. K., Sarkhel, B. C. (2012). In goats, TALENs were first reported in fibroblasts to introgress SNP alleles that are responsible for fecundity in sheep (bone morphogenetic protein receptor type 1B, BMPR-IB; also known as Booroola fecundity, FecB) and muscle hypertrophy (callipyge, CLPG) into the goat genome using TALEN mRNA and oligonucleotide transfection (Tan et al., 2013). Eid, A., Alshareef, S., Mahfouz, M. M. (2018). (2016a). (2016a). The list of sheep and goat genes targeted by CRISPR systems is continuously growing. Mol. Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos. doi: 10.1093/hmg/ddq063, Jain, H., Singh, S., Kadam, M., Sarkhel, B. C. (2010). doi: 10.1007/s12033-015-9902-1, Tian, H., Luo, J., Zhang, Z., Wu, J., Zhang, T., Busato, S., et al. Rep. 6, 29855. doi: 10.1038/srep29855, Gupta, R. M., Musunuru, K. (2014). 29, 413–418. Genet. One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system. (2011). 123, 225–235. During the second wave of the development of these enabling technical tools, new and more advanced techniques emerged. (1999). The reported efficiencies of CRISPR/Cas9 in goat fibroblasts ranged from 9 and 70%, indicating the ability of CRISPR/Cas9 to efficiently work in the caprine system. 30, 2299–2306. (2013). Biochem. Mol. 64, 2933–2944. The rhA expression was roughly proportional to the transgene copy number. In an effort to provide an abundant source of rhA, a herd of transgenic cows expressing high levels of hA in their milk has been developed. Zool. Biosci. 27, 44–51. Credit: Manu Palomeque/Alamy. for the cost-effective production of high-value pharmaceuticals in the blood or milk of transgenic sheep or cows. Transgenic technologies form today the core of ‘interventional genetics’, a key methodology for analyzing and interfering with gene function in whole animals. Cell. (2017). Nature 315, 680–683. The simplicity of TALENs over ZFNs has extended the application of gene editing and facilitated the generation of organisms as well as cell lines with specific genetic alterations. 29, 1500–1507. Mol. doi: 10.1007/s12010-008-8346-5, Xiong, K., Li, S., Zhang, H., Cui, Y., Yu, D., Li, Y., et al. doi: 10.1111/febs.14520, Zhang, J., Li, L., Cai, Y., Xu, X., Chen, J., Wu, Y., et al. 18, 19–23. 12, 3363–3374. Recombinant human antithrombin expressed in the milk of non-transgenic goats exhibits high efficiency on rat DIC model.

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