Transgenic Animals


Transgenesis implies that a foreign deoxyribonucleic acid (DNA) fragment is introduced into the genome of a multicellular organism and transmitted to progeny. Transgenesis, therefore, differs from gene transfer into cultured cells (transfection) or into the somatic cells of a patient (gene therapy). The foreign DNA can integrate randomly in host genome leading to gene addition or in a targeted manner making it possible precise endogenous gene inactivation or replacement. Transgenesis allows transferring genes from any source in a single generation. Transgenesis is, therefore, complementary to spontaneous mutations, which take place at each reproduction cycle or which are experimentally induced by mutagenic compounds or irradiation. These conventional approaches must be followed by appropriate selection of animals. Transgenesis has become an essential tool to study gene function especially in the medical and pharmaceutical fields. Improvements of breeding and food are also in course. Recent techniques based on the targeted gene modification make it much easier specific gene knockout and gene replacement in animals. The CRISPR‐Cas9 system is the most currently used system.

Key Concepts

  • Transgenesis is more precise and more diverse than conventional selection.
  • Transgenesis and conventional selection are complementary.
  • Transgenesis is an essential tool to study gene action and control.
  • Cloning is presently intensively used to generate transgenic farm animals.
  • Stem cells are currently used for random and targeted gene integration.
  • Targeted gene integration using double‐strand genomic DNA break provides researchers with exceptionally efficient new tools.
  • Conventional targeted gene transfer is limited by its low efficiency.
  • SiRNAs are efficient tools to inhibit gene expression at the mRNA level.
  • The available tools to generate transgenic animals offer the possibility to develop more relevant biological models and to prepare safer food.
  • Animal behaviourists must participate in conservation planning to protect the future of biodiversity.

Keywords: gene addition; gene replacement; cloning; microinjection; animal models; animal bioreactors; animals as organ donors; improved farm animals

Figure 1. Effect of evolution and transgenesis on genome modification. The classical genetic selection relies on the recombination of homologous chromosomes during gamete formation and the random distribution of parental genes to progeny. Transgenesis provides organisms in one generation with exogenous genes having known and potentially useful properties.
Figure 2. The generation of transgenic animals by gene microinjection. The embryos obtained by superovulation or by in vitro fertilisation receive the foreign genes and are developed in foster mothers. Transgenes are detected and transmitted to progeny by normal reproduction. PCR, polymerase chain reaction.
Figure 3. Different methods to generate transgenic animals: (1) DNA (deoxyribonucleic acid) transfer via direct microinjection into pronucleus or cytoplasm of embryo; (2) DNA transfer via a transposon: the foreign gene is introduced in the transposon that is injected into a pronucleus; (3) DNA transfer via a lentiviral vector: the gene of interest introduced in a lentiviral vector is injected between the zona pellucida and membrane of the oocyte or the embryo; (4) DNA transfer via sperm: sperm is incubated with the foreign gene and injected into the oocyte cytoplasm for fertilisation by intracytoplamic sperm injection (ICSI); (5) DNA transfer via pluripotent or multipotent cells. The foreign gene is introduced into pluripotent cell lines (ESC (embryonic stem cell) lines established from early embryo or iPS, induced pluripotent cells obtained after dedifferentiation of somatic cells) or into multipotent cell lines (EGC (embryonic gonad cell) lines established from primordial germ cells of foetal gonads). The pluripotent cells containing the foreign gene are injected into an early embryo to generate chimaeric animals harbouring the foreign gene DNA. The multipotent EGCs containing the foreign gene are injected into chicken embryos to generate gametes harbouring the transgene. In both cases, the transgene is transmitted to progeny; (6) DNA transfer via cloning: the foreign gene is transferred into a somatic cell, the nucleus of which is introduced into the cytoplasm of an enucleated oocyte to generate a transgenic clone. Methods 1–4 allow traditionally random gene addition, whereas methods 5 and 6 allow random gene addition and targeted gene integration via homologous recombination for gene addition or gene replacement including gene knockout and knockin. The use of engineered endonucleases to cut both DNA strands makes it possible targeted gene knockin and knockout in one cell embryos. Modified from Houdebine © Springer‐Verlag.
Figure 4. The transmission of a mutation by the cloning technique. The foetal cells in which gene addition or replacement occurred are used to generate living embryos after transfer into enucleated oocytes. The mutation is transmitted to progeny.
Figure 5. The transmission of a mutation by the generation of chimaeric animals. The pluripotent embryonic cells in which gene replacement occurred are transferred into a recipient embryo and participate in its development. The mutation can be transmitted to progeny.
Figure 6. The mechanisms leading to the random integration of a foreign gene into an animal genome. The foreign DNA sequences recognise short and partially homologous regions of the genome. Reparation mechanisms integrate the foreign DNA. Before integration, a homologous recombination mechanism generates polymers of the foreign gene organised in tandem.
Figure 7. The experimental protocol leading to specific gene replacement. A gene construct containing two long regions strictly homologous to the targeted host gene and containing a foreign DNA region is transferred to cells. The homologous sequences recombine, and the targeted gene is replaced by the foreign gene. The cells in which gene replacement occurred are saved by double selection (not shown here).
Figure 8. Schematic representation of the CRISPR‐Cas9 system. crRNA is the RNA, which target the position of the Cas9 endonuclease; trancRNA is a fusion of crRNA and the small RNA associated to Cas9; NGG and NCC are signals for the binding of the crRNA to targeted site in genomic DNA.
Figure 9. Mechanisms of action of double breaks in genomic DNA. In the absence of foreign DNA the repair mechanisms of the cell bridge the gap by adding nucleotides (NNN…) randomly and with high efficiency. This mechanism that is known as nonhomologous end joining (NHEJ) is a targeted knockout. In the presence of DNA with sequence similar to this of the targeted genomic site the foreign DNA is used as a template inducing a homologous recombination. This event is a knockin allowing targeted gene addition or allele replacement.


Ayares D (2010) Genetic modification of pigs for xenotransplantation. Meeting UC Davis Transgenic Animal Research Conference VII. Tahoe City, USA. Transgenic Research 19: 143.

Boland MJ, Hazen JL, Nazor KL, et al. (2012) Generation of mice derived from induced pluripotent stem cells. Journal of Visualized Experiments 69: e4003. DOI: 10.3791/4003.

Cyranoski D (2015) Super‐muscly pigs created by small genetic tweak. Nature 523: 13–14. DOI: 10.1038/523013a.

Daniel‐Carlier N, Sawafta A, Passet B, et al. (2013) Viral infection resistance conferred on mice by siRNA transgenesis. Transgenic Research 22: 489–500. DOI: 10.1007/s11248-012-9649-4.

Fahrenkrug SC, Blake A, Carlson DF, et al. (2010) Precision genetics for complex objectives in animal agriculture. Journal of Animal Science 88: 2530–2539. DOI: 10.2527/jas.2010-2847.

Gabriel R, Lombardo A, Arens A, et al. (2011) An unbiased genome‐wide analysis of zinc‐finger nuclease specificity. Nature Biotechnology 29: 816–823. DOI: 10.1038/nbt.1948.

Gannon F (2007) Animal rights, human wrongs? Introduction to the talking point on the use of animals in scientific research. EMBO Reports 8 (6): 519–520.

Gantz VM, Jasinskiene N, Tatarenkova O, et al. (2015) Highly efficient Cas9‐mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proceedings of the National Academy of Sciences of the United States of America 112 (49): E6736–E6743. DOI: 10.1073/pnas.1521077112.

Gao Y, Wu H, WangY LX, et al. (2017) Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off‐target effects. Genome Biology 18: 13. DOI: 10.1186/s13059-016-1144-4.

Garrels W, Mates L, Holler S, et al. (2011) Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome. PLoS One 6: e23573.

Hammond A, Galizi R, Kyrou K, et al. (2016) A CRISPR‐Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology 34 (1): 78–83. DOI: 10.1038/nbt.3439.

Han JY (2009) Germ cells and transgenic chicken. Comparative Immunology, Microbiology & Infectious Diseases 32: 61–80.

Hauschild‐Quintern J, Petersen B, Queisser AL, et al. (2013) Gender nonspecific efficacy of ZFN mediated gene targeting in pigs. Transgenic Research 22: 1–3.

Houdebine LM (2009b) Methods to generate transgenic animals. In: Engelhard M, Hagen K and Boysen M (eds) Genetic Engineering in Livestock New Applications and Interdisciplinary Perspectives Series: Ethics of Science and Technology Assessment, vol. 34, pp. 31–48. Berlin: Springer‐Verlag.

Ledford H (2015) Salmon approval heralds rethink of transgenic animals. Nature 527: 417–418. DOI: 10.1038/527417a.

Li T, Huang S, Jiang WZ, et al. (2010) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA‐cleavage domain. Nucleic Acids Research 39 (1): 359–372. DOI: 10.1093/nar/gkq704.

Lillico S, Vasey D, King T and Whitelaw B (2011) Lentiviral transgenesis in livestock. Transgenic Research 20: 441–442.

Lillico SG, Proudfoot C, Carlson DF, et al. (2013) Live pigs produced from genome edited zygotes. Scientific Reports 3: 2847. DOI: 10.1038/srep02847.

Lyall J, Irvine RM, Sherman A, et al. (2011) Suppression of avian influenza transmission in genetically modified chickens. Science 331: 223–226.

Meyer M, Hrabé de Angelis M, Wursta W and Kühn R (2010) Gene targeting by homologous recombination in mouse zygotes mediated by zinc‐finger nucleases. Proceedings of the National Academy of Sciences of the United States of America 107: 15022–15026.

Moreira PN, Pozueta J, Perez‐Crespo M, et al. (2007) Improving the generation of genomic‐type transgenic mice by ICSI. Transgenic Research 16: 163–168.

Mussolino C and Cathomen T (2013) RNA guides genome engineering. Nature Biotechnology 31 (3): 208–209.

Nature Methods (2012) A special issue containing six reviews on ZFN and TALEN. Nature Methods 9: 28–34.

Perkel JM (2013) The new genetic engineering toolbox. BioTechniques 54: 185–188. DOI: 10.2144/000114007H.

Petersen B, Carnwath JW and Niemann H (2009) The perspectives for porcine‐to‐human xenografts. Comparative Immunology, Microbiology and Infectious Diseases 32: 91–105.

Ravindran S (2016) Eliminating CRISPR‐Cas9's off‐target effects. Biotechniques.‐CRISPR‐Cas9s‐Off‐target‐Effects/biotechniques‐362580.html#.WO4ksaLfPDc (accessed 13 Jan 2016).

Regalado A (2014) On the Horns of the GMO Dilemma. MIT Technology Review, September 2.‐the‐horns‐of‐the‐gmo‐dilemma/ (accessed 13 Dec 2017).

Rémy S, Tesson L, Menoret S, Usal C, et al. (2010) Zinc‐finger nucleases: a powerful tool for genetic engineering of animals. Transgenic Research 19: 363–371.

Reyon D, Tsai SQ, Khayter C, et al. (2012) FLASH assembly of TALENs for high‐throughput genome editing. Nature Biotechnology 30: 460–465. DOI: 10.1038/nbt.2170.

RNAi: Multi‐author Review (2009) Insight: RNA silencing. Nature 457 (7228): 395–434.

Scolari F, Siciliano P, Gabrieli P, et al. (2011) Safe and fit genetically modified insects for pest control: from lab to field applications. Genetica 139: 41–52.

Tabashnik BE, Sisterson MS, Ellsworth PC, et al. (2010) Suppressing resistance to Bt cotton with sterile insect releases. Nature Biotechnology 28: 1304–1307.

Urschitz J, Kawasumi M, Owens J, et al. (2010) Helper‐independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proceedings of the National Academy of Sciences of the United States of America 107: 8117–8122.

Van Eenennaam AL and Muir WM (2011) Transgenic salmon: a final leap to the grocery shelf? Nature Biotechnology 29: 706–710.

Van de Lavoir M, Diamond HH and Leighton PA (2006) Germline transmission of genetically modified primordial germ cells. Nature 441: 766–769.

Vàzquez‐Salat N, Salter B, Smets G, et al. (2012) The current state of GMO governance: are we ready for GM animals? Biotechnology Advances 30: 1336–1343.

Vàzquez‐Salat N and Houdebine LM (2013) Will GM animals follow the GM plant fate? Transgenic Research 22: 5–13. DOI: 10.1007/s11248-012-9648-5.

Whitworth KM, Rowland RR, Ewen CL, et al. (2016) Gene‐edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature Biotechnology 34 (1): 20–22. DOI: 10.1038/nbt.3434.

Woods IG and Schier AF (2008) Targeted mutagenesis in zebrafish. Nature Biotechnology 26: 650–651.

Yong HY, Hao Y, Lai L, et al. (2006) Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm. Molecular Reproduction and Development 73: 595–599.

Further Reading

ACTA (2010) Genetically Modified Insects: What Next? London: mi2g Ltd..

Bruce A (2017) Genome edited animals: learning from GM crops? Transgenic Research 26: 385–398. DOI: 10.1007/s11248-017-0017-2.

Giraldo P, Rival‐Gervier S, Houdebine LM, et al. (2003) The potential benefits of insulators on heterologous constructs in transgenic animals. Transgenic Research 12: 751–755.

Houdebine LM (2003) Animal Transgenesis and Cloning. Chichester: John Wiley & Sons, Ltd.

Houdebine LM (2009a) Production of pharmaceutical proteins by transgenic animals. Comparative Immunology, Microbiology & Infectious Diseases 32: 107–121.

Houdebine LM (2009c) Design of expression cassettes for the generation of transgenic animals (including insulators). In: Anegon I (ed.) Rat Genomics: Methods in Molecular Biology, vol. 597, pp. 55–69. New York: Humana Press, a part of Springer Science + Business Media, LLC2010. Dordrecht: Humana Press, a part of Springer Science + Business Media, LLC2010, Heidelberg: Humana Press, a part of Springer Science + Business Media, LLC2010 and London: Humana Press, a part of Springer Science + Business Media, LLC2010.

Houdebine LM (2011) Production of human polyclonal antibodies by transgenic animals. Advances in Bioscience and Biotechnology 2: 138–141.

Houdebine LM (2012) A review on the book. In: Pease S and Saunders TL (eds) Advanced Protocols for Animal Transgenesis: An ISTT Manual Springer Protocols. Berlin: Springer‐Verlag, 2011, Hardcover, 684 pp, ISBN‐13: 978–3642207914. Transgenic Research 21: 1143–1148. doi: 10.1007/s11248-011-9583-x.

Houdebine LM, Lema MA and Burachik M (2012) Generation of Genetically Modified Animals In Collection of Biosafety Reviews, vol. 7, p. 93. Trieste: International Centre for Genetic Engineering and Biotechnology.

Niemann H, Kuhla B and Flachowsky G (2011) Perspectives for feed‐efficient animal production. Journal of Animal Science 89: 4344–4463. DOI: 10.2527/jas.2011-4235.

Prather RS, Shen M and Dai Y (2008) Genetically modified pigs for medicine and agriculture. Biotechnology and Genetic Engineering Reviews 25: 245–266.

Tan W, Carlson DF, Lancto CA, et al. (2013) Efficient non meiotic allele introgression in livestock using custom endonucleases. Proceedings of the National Academy of Sciences of the United States of America 110: 16526–16531.

Wall RJ, Laible G, Maga E, Seidel G and Whitelaw B (2009) Animal Productivity and Genetic Diversity: Cloned and Transgenic Animals – Animal Agriculture's Future Through Biotechnology, Part 8. Issue Paper, no. 43, 16 pp. Council for Agricultural Science and Technology.

Wells KD (2016) History and future of genetically engineered food animal regulation. Transgenic Research 25 (3): 385–394. DOI: 10.1007/s11248-016-9935-7.

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Houdebine, Louis M(Feb 2018) Transgenic Animals. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000990.pub4]