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Pronuclear Injection
Pro-nuclear Injection to make Transgenic Mice
Transgenic mice are generated through pro-nuclear injection with DNA or RNA. Transgenic mice are generated to:
- Affect expression of a specific gene in a positive or negative fashion
- Assess gene regulatory elements in an in vivo environment
- Produce spatial and temporal expression patterns of gene expression, often related to cre or frt - mediated recombination.
The LRTGT guarantees production of three founder mice, unless the transgene or genetic manipulation is lethal at an embryonic age. If such is the case, we will work with the investigator at an additional charge to identify embryonic mice carrying the transgene.
To undertake pronuclear injections, the LRTGT requires a detailed plasmid map of the transgenic DNA construct as well as a protocol for isolating the transgene from all bacterial sequences. This should be documented by a picture showing digested plasmid with the transgene removed. In addition, the investigators must provide evidence that they have developed a PCR-based strategy for detecting a single copy, inserted transgene on genomic DNA.
Finally, the investigators should provide some expectations regarding the possible outcomes for the transgenic lines, especially regarding the potential for embryonic or neonatal lethality. Similarly, for RNA injections, we will need evidence of RNA purity and integrity and evidence of a working genotype strategy to identify desired mutations. This is essential to begin a project.
Using this DNA or RNA, 150 embryos will be processed for pro-nuclear injection and implanted back into female mice. From this procedure, we expect to obtain 40 potential founder pups. The investigator will be informed on the number of pups and any unusual phenotypes.
Two weeks after delivery, tail biopsies will be obtained and DNA isolated. We will provide DNA to investigators for genotype analysis. Once pups are weaned, we will either ship mice to the investigator or maintain the potential founders for two weeks at the investigator’s expense. Once the founder mice have been delivered, we will provide a detailed report regarding the project. We also request follow up reports from the investigators.
