Pronuclear injection is the most widely used method to generate transgenic mice. The C57BL/6 and FVB mouse strains used for transgenic injection are provided and inbred by ENVIGO or by Charles River.
DNA is directly microinjected into the male pronucleus of fertilized oocytes. The customer should provide at least 30 micrograms of the DNA construct to be purified and CFCM staff purifies the DNA. CFCM microinjects the transgene into 150 fertilized eggs. Embryos are transferred into foster mothers and pregnancies are monitored. The investigators then receive tail biopsies from 14 days old mice for genotyping; and, for transgenic embryos job, the customer receives pregnant mothers.
CFCM requires investigators to be able to screen transgenic mice before sending to CFCM the construct.
How to screen for transgenic mice
Transgenic mice may be identified by Polymerase Chain Reaction and/or by Southern blot analysis. In the first case, users should synthesize primers specific for the transgene and set up PCR conditions using serial dilutions of the transgene DNA with genomic DNA in order to mimic conditions in putative transgenic mice. In the second case, appropriate restriction enzymes should be chosen as well as a probe. The probe should be tested on transgene DNA mixed to genomic DNA (more information can be found in Manipulating the Mouse Embryo, CSHL-PRESS).