Microinjection or electroporation (Rats)
Information about pronuclear or cytoplasmic injection of DNA, RNA or CRISPR reagents to generate transgenic rats
Transgenic rats can be generated by pronuclear or cytoplasmic injection or electroporation of DNA, RNA or CRISPR reagents into fertilized eggs. Fertilized eggs are obtained from superovulated female donor rats that are mated with fertile (stud) males. The injection mix is then either electroporated or microinjected into either the cytoplasm or the pronuclei of each egg. Injected/electroporated eggs are usually cultured overnight to the 2-cell stage and then transferred to the oviducts of pseudopregnant “recipient” females (recipients are generated by mating females with vasectomized males so the females do not produce any fertilized embryos of their own).
It is essential to provide CTC with the highest quality DNA, RNA or CRISPR injection mix for injecting into rat zyogtes.
Researcher’s responsibilities during process:
- Complete the ‘Request to Produce Transgenics Form’ and return it to the CTC manager (please see below).
- Design and provide evidence of a screening strategy for founders (i.e. PCR).
- If injecting DNA, prepare DNA as per CTC guidelines (please see below). It is the researcher’s responsibility to make sure the DNA construct has been purified correctly and the concentration determined accurately. Inaccuracies in the concentration of as little as 2-fold can have significant effects on the production of transgenic founders.
- Deliver pre-prepared reagents (DNA, RNA or CRISPR mix) to be injected to the CTC Facility any time prior to the date of injection or latest by 10am on the injection day. Alternatively, we can arrange for pick-up of injection mixes from any University of Edinburgh location and delivery to the CTC facility before the injection day.
- Screen the DNA from founders to confirm genetically altered (GA) offspring.
- Provide feedback to CTC regarding the number of GA animals generated once genotyping of pups has been performed (please see below).
CTC’s responsibilities during process:
- CTC will provide vasectomized and organize the purchase of female rats and studs on behalf of the researcher.
- CTC staff will communicate with the researcher and arrange suitable dates for injection/electroporation sessions.
- More than one injection/electroporation session may be required.