Transgenic Animals

Abstract

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 2009b © 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.
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Houdebine, Louis M(Feb 2018) Transgenic Animals. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000990.pub4]