DNAVision provides in collaboration with BV Transgenic Services (Gosselies, Belgium) proposes a wide range of transgenic services involving the development of animal models from DNA construct to phenotype analysis. Our non-exhaustive panel of services include:
Fully equipped laboratories and know-how are available to support your transgenic activities.
The health status of laboratory animals may significantly influence the outcome of experimental procedures. It is generally accepted that the target microbiological status is that specified within the FELASA Recommendations (FELASA, 2002). In this way, the reporting of microbiological status is standardised allowing animals to be exchanged between laboratories and breeders in the confidence that known microbial adventitious agents will not be inadvertently introduced.
Development and maintenance of genetically altered animals under such stringent microbiological conditions may be complex and may require a highly controlled environment, which is expensive to maintain. The introduction of a new line into such an environment demands strict precautions in order to avoid the entry of pathogens into the facility.
Embryo transfer is a reliable method of introducing new lines into an animal facility based on the experience that embryos in the uterus are in a pathogen free environment. Animals (heterozgotes or homozygotes) are received into our Quarantine area and are mated according to your instructions. Pre-implanted embryos are removed, washed in sterile medium and transferred into the uterus of an adventitious-pathogen-free pseudo-pregnant female. The resulting pups are, therefore, free from pathogenic organisms at birth. Subsequent microbiological monitoring of the pups and/or foster mothers is carried out to verify the status. The resulting animals are returned to you at approximately 6 weeks of age.
The number of genetically altered lines developed and maintained for research in animal facilities continually increases. Laboratories often develop their own strains, genetically modified for specific purposes. The high value of these strains often results in their continued maintenance, even though they may not be utilised for long periods. Such maintenance may produce excess animals, and is also financially expensive.
Cryopreservation of valuable strains as frozen embryos provides a less expensive, more ethical and efficient means of maintaining them. Cryopreservation may also provide a ‘back-up’ for stocks and strains held within the animal facility and a source of animals free from microbiological and genetic contamination.
Embryos from a pregnant female are removed at the 8-cell stage of development and frozen in a cryoprotectant medium. They are then frozen for long term storage in liquid nitrogen. When required by the customer, the strain can be revitalized by thawing the embryos and re-implanting them into a pseudo-pregnant female. This is only carried out after the vitality and viability has been checked by in vitro culture.
Animals (heterozgotes or homozygotes) are received into our Quarantine area and mated to obtain embryos for freezing at the 8-cell stage. Between 200 and 300 embryos are frozen in cryotubes, each containing about 40 embryos. During the process, various tests are carried out to verify the quality of the freezing procedure. A number of the embryos are cultured in vitro at the following stages of the process: before addition of the cryoprotectant, before freezing and after one week of crypreservation in liquid nitrogen. Embryo culture progresses until the blastocyste stage.
The process of embryo freezing can be combined with rederivation.
Genetically modified mice can be developed in various ways. One of these is to create a new strain by homologous recombination. A targeting vector that contains the mutation to be introduced into the mouse genome is constructed. It is then introduced into mouse ES stem cells by electroporation. After culture and selection for the genomic insertion of the construct into the genome of the cells, recombinant clones can be identified.
These recombinant clones are microinjected into mouse blastocysts and the modified embryos are re-implanted into a pseudo-pregnant female. The ES stem cells are able to colonize the embryo and participate in its development. Resulting pups may show coats of mixed coat colours from the ES cells and the host embryo. Such animals are called chimeras. These chimeras are able to transmit the mutation to their descendents and so become founders of a new genetically modified strain.
Genetically modified mouse ES stem cells are available in the scientific community and can be easily shipped. We offer a service to microinject these ES cells into mouse blastocysts to generate your chimeras.
We will request you to supply ES cells, together with known health status and karyotype data.
The clone is amplified and part of it frozen in liquid nitrogen. Cells are injected until at least 3 chimeras are obtained, with a good percentage of chimerism, or until the injection of 150 blastocysts. Those chimeras returned will be at the same high health status of the mothers and can be used for further breeding.
Laboratory animals are used as research models for human diseases. Many strains have been developed and maintained and this number will continue to increase every year. The importance of the genetic background in experimental procedures is well established and well documented and takes a central place in all research using laboratory animals.. It may be argued that the genetic status of laboratory animals is as important as health status to the outcome of experimental procedures.
DNAVision and BV Transgenic Services have established genetic monitoring programmes to facilitate the genetic characterisation of your strains. This will allow you to determine the genetic identity and degree of inbreeding of your strain relative to known strains, or to detect potential genetic contaminations in your colony. Using microsatellite polymorphism between strains we are able to perform the profiling of your strains and answer questions regarding genetic identity of these strains before they are used in experiments.
Our two programmes:
A defined and validated panel of 20 microsatellite markers allows rapid characterisation of your strain. The panel is sufficient to discriminate between all strains of inbred mice, and will differentiate between closely related strains such as C57BL/6 and C57BL/10.
The Quick Scan procedure can also be used as a first approach in the case of a suspected genetic contamination.
Specific microsatellites or SNPs can be added to the process on request.
Defined and validated panels of 100 microsatellites, separated by 15 cM along the genome, are used in the Genome Scan. The panels are sufficient to establish the degree of inbreeding of the strains. The Genome Scan is not only useful in determining when a backcrossing procedure has reached completion ,but also can be used to localise the locus involved in a phenotype.
The panel can also identify genetic contamination events, even if they occurred some time previously. In this way, the method provides assurance of the inbred status of the inbred strains used in the experiment, and that experimental observations are due only to the specific mutation. Specific microsatellites or SNPs can be added to the process on request.
For both programmes, the procedure can utilise DNA extracted for tail-snips or ear slices). A full report together with analysis will be provided at the end of the procedure.
The development of transgenic animals produces models with unsuitable or mixed genetic background making them inappropriate for biomedical studies. Historically, to resolve this problem, mutant animals are backcrossed onto wild type animals which have the desired inbred genetic background. In the backcrossing process, the presence of the mutation is monitored and it is expected that after 10 generations (from F1 to N10) the genetic background is inbred and that the mutation is fully integrated (congenic mice). 2 - 3 years are necessary to complete the process; in today’s competitive research arena, such a delay may be unacceptable.. The purpose of the speed congenic procedure is to significantly decrease the time required to transfer a mutation of interest from one genetic background (donor strain) to another (host strain - typically inbred).
Applying the speed congenic procedure, the transmission of the targeted mutation is first monitored as for backcrossing, but in addition the genome is scanned for polymorphic micro-satellites. Using this technique, only 5 generations (from F1 to N5) are required to obtain a congenic mouse. The process is complete in 10 to 14 months and the genetic background is at least 99% of the host strain genome.
For the animal strains involved in each project, a panel is designed consisting of 100 polymorphic microsatellites located throughout the genome and separated by 15 cM. The genome of 5 heterozygous mice per generation is scanned and the most suitable with respect to host genome content is used as founder for the next generation. The choice of the microsatellite panel is based on available databases and takes into account chromosomal location of the mutation. In the case of unavailability of a panel covering the entire genome the microsatellite analysis can be combined with specific SNP genotyping.
BV Transgenic Services is able to provide colony maintenance to microsatellite analysis.
Animals are maintained in flexible film isolators or IVCs. Tissue samples, usually tail snips, are treated for DNA extraction and analysed by PCR to detect the presence of the mutation according to the client protocol, and heterozygous animals are analysed for their homozygosity in host strain microsatellites. A significant portion of the genome of the two most suitable N5 animals is then scanned to monitor the quality of the speed congenic procedure.
Finally, at least these two heterozygous animals of high microbiological status will be returned to the customer in order to set up the new congenic colony. At each generation, an intermediary report is sent to the customer summarising the results of the microsatellite analysis, and the founder chosen for the next generation.
The significant advantage of the speed congenic procedure is the time saved (18 – 24 months) when compared with traditional backcrossing techniques.
By analysis of the genome at each generation, the most suitable founder for the next generation can be verified. In the traditional backcross technique, there are no such monitoring steps, which may lead to unexpected and undesirable outcomes. Our goal is to provide genetically verified models for your research.
What we need from the customer: