Pronuclear Injection Service

Pronuclear Injection is the technical term for the process of making a transgenic mouse.  More specifically, it is the objective of the microinjection process. That is, the injectionist uses very fine glass microinjection needles to inject picoliters of injection solution into one of the pronuclei in the fertilized egg prior to their fusion to make a diploid nucleus. The injection solution contains 1-2 nano grams of prepared transgene per micro liter. Transgenic DNA is typically either plasmid-based or BAC-based. When using plasmid DNA our facility requires the method for separating the vector backbone from the actual expression cassette that will be the transgene.  Typically, one or two restriction enzymes are used for this process. The Core prefers to make fresh preparations of the transgene prior to each injection session. Generally, the transgene and the vector backbone are separated using gel electrophoresis and the transgene is purified with a Qiagen kit and suspended in endotoxin-free water.

BAC based transgenes are typically injected as uncut vectors because the BAC contains sizable flanking regions which insulate the transgene expression from the BAC vector backbone effect and any influence of the flanking genomic sequences. BAC preps are also purified and resuspended in polyamine solutions to coat and stabilize these sizable DNA elements prior to pronuclear micro injection. 

The transgenic core only uses B6 X SJL F2 eggs for pronuclear injection.  For those investigators who want their transgenes on and inbred background, we suggest the use of marker assisted back breeding (see Moore et al. Immunol Today. 1997 Oct;18(10):472-7).Micro injected eggs are placed in culture overnight to determine which embryos retain developmental competence. A very small percentage of embryos are damaged by the transgene injection process. The surviving two-cell embryos are implanted into pseudo pregnant recipients and produce mouse pups in about 3 weeks.  When the mice are weaned typically around 3 weeks of age, they are transferred to the investigators account and tail clips are taken and PCR-genotyping is done by the Core to identify founders. The progress of a transgenic project is recorded on the pronuclear project status page on a weekly basis. The project status page will display projects in several categories as explained on that web page.

Each transgenic founder is a unique animal line and should be treated as such. Transgenes nearly always insert as concatemers of varying numbers and at random sites.  It is possible that the transgene will not insert into the genome until after the first cell division resulting in a mosaic mouse that is PCR positive but may not transmit the transgene though the germ line. Transgene expression is not synonymous with transgene copy number.  Many observations indicate that the flanking sequences of the transgene insertion sites can have very dramatic effects on the expression of the transgene. Yet another complication is that it is possible to get two different transgene insertion sites in one founder. This is easily detectable by doing a Southern blot after cutting genomic DNA with enzymes that do not cut within the transgene. The blot is probed with the transgene and multiple bands indicate multiple insertion sites.  As these sites segregate from each other during meiosis it is possible to get different mice that are transgene positive.  In a worse case, one might retain the non expressing, transgene positive mouse line and loose the transgene expressing mouse line. Therefore, this recommendation to do a Southern blot is very important. Yet another process can occur during transgene insertion that may confound the analysis of the phenotype observed in the presence of the transgene. This is the distinct possibility of insertional mutagenesis of an endogenous gene. Rarely will a transgene insert into an exon.  However, all transgenes contain poly A signals that terminate transcription. The artificial placement of a poly A signal in the intron of a gene will usually render the gene non functional.  Due to these various scenarios, careful record keeping of your transgenic mice is essential. Furthermore, multiple mice must show the identical phenotype to rule out any insertion site-mediated phenotype.