Introduction and considerations
Establishment of a transgenic line is a relatively fast approach to investigate the function of a particular gene. Well-established protocols for all steps involved and the relative low cost for generation of a transgenic line makes pronuclear injection an attractive option.
With regular pronuclear injection, the injected DNA-construct will be randomly integrated in the genome. The number of integrated copies of the construct ranges from one to more than hundreds, where multiple copies of the construct are integrated in tandem at a single chromosomal locus (occasionally integrated in a few loci). Methods have been developed also for site-specific integration of the construct. The latter strategy must be planned individually for each project.
Although pronuclear injection is a powerful technique, it is important to understand the limitations. The construct successfully inserted in a founder may not be expressed in the desired manner. It is not always possibly to discern the reason behind a lack of the expected expression. The construct may e.g. be inserted in a transcriptionally inactive region or be recognized as foreign DNA and inactivated by methylation. Further, one or more components in the construct may not be optimal for in vivo expression. Such issues may be caused by non-optimal codon usage, a non-optimal promoter region used, or untranslated regions remaining in the construct. Still, if the construct is well designed and passes recommended control experiments in cell culture, one has a fair chance to get the desired transgenic animal amongst a few generated founders.
Description of the service
- A DNA construct, gel purified to remove vector backbone, is injected into the pronucleus of 1-cell stage embryos.
- Following injection, embryos are transferred into oviducts of a pseudopregnant foster mother where they develop to term. Offspring is born approximately 20 days later.
- Mice that have the transgene integrated in their genome can be identified by PCR. This step is normally performed by the customer.
Requirements and time-frame
- Construct: The customer must deliver a satisfactory designed construct. It is strongly advised to verify in vitro that the construct expresses the insert(s). The construct should contain at least one intron segment to ensure efficient mRNA processing and stability.
- NTS routinely injects into C57BL/6N, but the construct may be injected into embryos collected from mice of various genetic backgrounds.
- NTS will inject the construct into 200 embryos. To increase the success-rate for each project, injections will be distributed on 3-4 different injection days within a 3-4 weeks period. The customer can expect delivery of >30 offspring.
- Customer will receive biopsies and will be responsible for verification of positive founders.
- Project lasts typically for 10-15 weeks.
Technical service performed by NTS
- NTS is responsible for the animal procedure permit (FOTS application).
- Planning and coordination of the project.
- Restriction digestion, gel purification and dilution of the construct in a buffer suitable for injection.
- Superovulation of females, mating overnight, dissection of oviducts and collection of embryos.
- Injection of the construct into minimum 200 embryos.
- Implantation of embryos into pseudopregnant foster mothers.
- Separation of offspring and collection of ear biopsies.
- Customer will receive biopsies in house. If personal pick-up is not an option, transport will be charged customer.
- NTS expects confirmation of genotyping within a reasonable time (~1 week).
Costs cover the following
- Purchase and transport of donor females from vendors.
- Hosing of vasectomized males and foster mothers.
- Housing of males for mating with donor females for 4 weeks.
- Up to four rounds of injection or >200 embryos injected (whatever comes first).
- Confirmed birth of 30 offspring and delivery of 3 or more positive founders.
- Housing of offspring until 6 weeks of age.
Service Fees
Estimated costs for the above service can be found on our Service Fees page.