Central Transgenic Core

Microinjection

Information about pronuclear or cytoplasmic injection of DNA, RNA or CRISPR reagents to generate transgenic mice

 

Transgenic mice can be generated by pronuclear or cytoplasmic injection of DNA, RNA or CRISPR reagents into fertilized eggs.

Fertilized eggs are obtained from superovulated female donor mice that are mated with fertile (stud) males. The DNA/RNA/CRISPR reagents are then microinjected into either the cytoplasm or the pronucleus of each zygote. Injected zygotes and un-injected controls are usually cultured overnight to the 2-cell stage and then transferred into 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 and CRISPR reagents for injecting into mouse zygotes. 

We have an excellent track record of producing transgenic mice and the development of CRISPR technologies has opened the door to faster and more efficient generation of transgenic models. CTC has successfully produced both knockout and knock-in transgenic lines using CRISPR.

Different genetic background strains can be used to generate new transgenic lines, and we have good success with both hybrids and pure C57BL/6J. Creation of new strains directly onto inbred backgrounds (i.e. C57BL/6J) can avoid time-consuming breeding programs to achieve a congenic background. Hybrid donors like B6CBAF1 offer the advantage of an increased embryo yield and generally yield more pups after microinjection due to increased robustness. 

 

Researcher’s responsibilities during process:

  • Complete the ‘Request to Produce Transgenics Form’ (see below) and return it to the CTC manager.
  • 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 reagents) 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 transgenic offspring.
  • Provide feedback to CTC regarding the number of transgenics generated once genotyping of pups has been performed (please see below).

 

CTC’s responsibilities during process:

  • CTC will seek approval of the Transgenic PPL holder as well as the NVS for generating the new line.
  • CTC will provide vasectomized and stud males and organise the purchase of female mice on behalf of the researcher.
  • CTC staff will communicate with the researcher and arrange suitable dates for injection sessions.
  • More than one injection session may be required.