Genetic Modifications

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Genetic Modifications

 

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Pronuclear Injection

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.

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Blast Injection

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.

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CRISPR

The Core provides consultation and design of recombinant DNA molecules for targeted mutagenesis in rodent models, generation of gene-targeting vectors by recombineering, design of screening protocols for mice and ES cell clones (PCR and Southern-blot), quantification of transgene copy number, and BAC recombineering

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Custom Cloning - Recombineering

Recombineering (recombination-mediated genetic engineering) is a novel and powerful method which enables fast and efficient construction of vectors to generate complex gene modifications in the mouse genome.

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Gene Targeting

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.

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Rederivation, Cryopreservation, IVF

Due to microbial infections, it can be necessary to rederive an existing mouse strain. 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. 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.