Pronuclear injection is a very common and well established method used to create transgenic mice. Fertilized eggs are collected at 0.5 dpc and one pronuclei is injected with a linearized DNA construct. The injected eggs are then transferred into the oviduct of pseudopregnant foster mice. On average, 15% of all pups born will have randomly integrated the injected DNA. This method is usually used for "gain-of-function" experiments.
ES CELL ELECTROPORATION
In order to modify endogenous genes, it is necessary to perform gene targeting. A vector containing a modified copy of the gene of interest is electroporated into mouse embryonic stem (ES) cells. The cells are subjected to drug selection, clones are picked and expanded and returned to the investigator for analysis. Homologous recombination is verified by either PCR or Southern blot analysis. Positive clones are expanded and injected into blastocysts.
In order to study the "loss-of-function" of any given gene it is necessary to replace the endogenous gene with a modified construct. This requires the creation of an embryonic stem (ES) cell line that is selected for homologous recombination. Positive ES cell clones are then injected into the blastocoel cavity of 3.5 dpc embryos which are then transferred into pseudopregnant foster mice. The injected ES cells co-mingle with the host embryo resulting in a chimeric offspring. Chimeras are easily identified by their coat coloring and then back-crossed to establish germline transmission.
In order to maintain heterozygous traits a tissue sample is obtained to confirm the genotype using PCR. At three weeks of age the animals are anesthetized and a tail biopsy taken. High-molecular weight genomic DNA is extracted and genotyped using PCR.
Due to microbial infections, it can be necessary to rederive an existing mouse strain. Three to ten week old females are superovulated and mated with a male of the infected strain. The following day the fertilized zygotes are harvested and transferred into clean pseudopregnant foster mice.
Cryopreservation of mouse germplasm is essential for anyone working with mouse mutant strains. It not only reduces the cost of animal care, saves space and facilitates sharing of valuable lines, it also minimizes the risk of strain loss due to contamination, genetic drift or disease. The facility strongly recommends embryo cryopreservation since the method works well with most stains. We also offer sperm freezing if desired.
In vitro fertilization (IVF) is performed to recover a mouse strain from frozen sperm. It can also be used to overcome breeding difficulties or rapidly expand an existing colony, since sperm from one male can potentially fertilize hundreds of zygotes.