The method used to produce a line of genetically engineered animals will essentially depend on the type of genetic modification required. However, when choosing the method, it is also necessary to take into account welfare issues and the possibilities of refinement to minimize harm to animal welfare and improve production efficiency.
The production of a genetically engineered animal must be preceded by confirmation that the line is not available elsewhere, since the unnecessary repetition of any production is a waste of animals. Section 1 of this document provides an overview of the methods used to produce genetically engineered animals.
Each method succinctly described is accompanied by the concerns for animal welfare raised as well as possibilities for refinement in order to reduce the number of animals required or improve the welfare of the animals used. The solutions proposed are supplemented by a discussion of other methods or improvements to those in use, revealing the refinement of gene expression control and reproduction techniques. For information on identification methods, current best practices for obtaining genetic material, and refinement possibilities for genotyping, see Alternatives for genotyping genetically engineered animals.
Techniques for producing animals modified by genetic engineering
Pronuclear injection is used to produce animals that express gene sequences under the control of a heterologous promoter. The promoter and the gene sequences can come from the same species or from different species.
This is a common technique in studies of the effects of overexpression or poor expression of certain genetic sequences. However, transgenes that express so-called negative constructs or interfering RNAs (small RNAi, microRNA, RNAi) can be used to produce knockout in organisms that cannot be targeted mutagenesis, as an alternative. to this approach to affect the functioning of multiprotein complexes or as a conditional mutagenesis technique when combined with directed recombinations or other technologies.
Opportunities for Refinement
The following methods can help solve problems related to random transgene integration: targeted transgenesis in embryonic stem cells, viral mediated gene transfer, transgenic artificial bacterial chromosomes (BAC) (section 1.4) and targeted transgenesis using zinc finger nucleases (ZFN) or effector nucleases of transcription activator type (TALEN), as well as refinement possibilities such as inducible and conditional transgenic expression methods, the use of insulators and rigorous selection of promoters. Other techniques can also address animal welfare issues used in the production of genetically engineered animals, such as non-surgical embryo transfer (NSET) and gene transfer by sperm. In addition, certain variables that may be linked to the design and preparation of a construction as well as the expression and transmission of the transgene should be taken into account when using pronuclear injection.
To reduce the risk of loss of transgene expression after several generations, subjects of the first generation of transgenics should be cryopreserved. It is then possible to recover the line in the event of loss of expression of the transgene, without having to create it again. Any additional founding lines not selected for experimental use should also be cryopreserved; it is useless to reproduce it over several generations.
Targeted transgenesis and mutagenesis in embryonic stem cells
Targeting refers to the insertion or deletion of genomic DNA sequences in a specific location. It is most often done by homologous recombination in mouse embryonic stem cells.
However, directed nuclease engineering has allowed targeting in mouse embryonic stem cells and in the embryos of various animals, including rats, rabbits and zebrafish. Site-specific recombinases (eg Cre and FLP) and site-specific integrases (eg PhiC31) are also used for targeting in embryonic stem cells and embryos. These methods allowing targeted transgenesis, that is to say integration of the transgene into a chosen locus, are adapted from targeted mutagenesis.
Since targeted transgenesis allows better control of genetic modification than pronuclear injection, it can be used to minimize the negative effects on the welfare of the animals produced.
Targeting genes in the embryo (e.g. using site-specific recombinases, nucleases or integrases) is one way to overcome the difficulties of pluripotency resulting from the use of embryonic stem cells , but the reduced efficiency with which embryonic genomes are modified may not lead to a reduction in the number of animals used.
To reduce the number of animals required for the production of target animal lines, screening should be done using parental embryonic stem cell lines with a high rate of germline success. Control can be optimized by the use of conditional knockout animals and inducible transgenes and can also limit the impact on the welfare of animals derived from biotechnology. Embryonic stem cells may have genomic or chromosomal abnormalities during cultivation (eg, aneuploidy) that can reduce their pluripotency and their rate of germ line transmission.
Chromosome counting helps determine whether the embryonic stem cells are euploid or aneuploid and can help select the clones most likely to contribute to the germ line. Therefore, counting should be done before microinjection of embryonic stem cells. According to the consensus among researchers in the field, the reference chromosomal propagation to produce chimeras corresponds to at least 50% of euploids during mitosis (evaluation of at least 20 transmissions).
However, some establishments only allow their use when a rate between 60% and 70% is reached. When all the clones descend from the same ancestor for a given directed mutation and the ploidy standards are not reached, it is possible to produce euploid subclones isolated by subcloning.
To do this, however, care must be taken to maintain the pluripotency of embryonic stem cells during subcloning and a second count of chromosomes should be carried out to verify the percentage of euploid cells. The use of co-isogenic embryonic stem cells (embryonic stem cells from the line targeted by animal testing) eliminates the need for backcrossing to achieve congenital status. Where possible, targeting should be done in coisogenic embryonic stem cells. In mice, methods of producing chimeras from exogamous host embryos (eg, aggregation or microinjection of an 8-blastomeric embryo) can reduce the number of embryo donors required, since females exogams produce more usable embryos than those produced by many common inbred lines.
It is also possible to increase the number of usable embryos by modifying the number of embryonic stem cells injected. Infectious contaminants can be a confounding factor in in vivo studies. Consequently, screening for pathogens is recommended as a best practice before microinjection of embryonic stem cells.
Viral mediated gene transfer (transgenesis by lentivirus)
Viral mediated gene transfer allows efficient gene transfer and sustained expression of transgenes. This method is useful when a rapid assessment of the phenotype associated with the expression or deregulation of a specific gene is required.
It ensures the efficient transfer of genes in cattle; the number of species suitable for certain studies is thus increased. Lentiviruses or RNAi constructs short enough to integrate easily into lentiviral vectors are alternatives to the production of knockout animals.
Bacterial artificial chromosomes (BAC)
The creation of transgenic animals with a BAC construct can protect the transgene of interest from endogenous promoters and other regulatory elements. This method can reduce positional effects, and the type of construct used very often includes all of the regulatory elements required for expression of the transgene of interest.