Preparation of DNA for Injection into Mouse Eggs

The vector sequences will have to be cut out from the transgene construct to release the insert (gene fragment) that you wish to have injected. This fragment must have a promoter, a start codon, a cDNA/genomic DNA/or reporter gene that you want to express, and a stop codon with a poly A region. Expression of cDNAs in transgenic mice may be further improved by inclusion of a heterologous intron between the promoter and coding region. There should be little or no vector present in the fragment to be injected. We recommend removing the entire plasmid backbone since plasmid sequences may influence the expression of your transgene. Although we have never injected fragments longer than about 15kb, fragments of pretty much any length may be microinjected (but the longer the fragment, the greater the care one should take to make sure it is not sheared during isolation).

Cut the plasmid (20 -100 µg, depending on the size of the insert) containing your transgene with the appropriate enzyme(s) to release the transgene insert. Run a minigel to check the digest. If the digest is okay, run the cut DNA in a 0.8% - 1.0% agarose gel in TAE. Use a preparative comb, or several wells of a standard comb. Stain briefly with Ethidium Bromide (it's good to stain after the gel-run to minimize exposure to light/EtBr) and cut the band of interest under long-wave U.V. (work very quickly to limit exposure to U.V.).

1. Extract the transgene fragment from the agarose band using “GeneClean” (BIO 101 Systems Products, Qbiogene). We prefer the “GeneClean Spin Kit” to the original GeneClean because the Spin Kit is less harsh (less likely to shear the DNA) and, further, does not contaminate the sample with glass (silica) beads, which will clog the microinjection needles and make it impossible to inject your sample. You may also use Qiagen Kits (Qiaex II and QIAquick) but the former kit may also introduce loose silica beads, while the latter kit is not suitable for fragments longer than 10kb. If you use loose-bead methods, please take time to clear your sample of beads - this can be done by successive centrifugation steps, with careful removal of the supernatant (leaving some behind so as not to pick up the beads).

2. Ethanol-NaAc precipitate the DNA solution.

3. Wash the DNA pellet several times with 70% ethanol and dry the pellet under vacuum. The importance of the washing and drying steps cannot be overemphasized, as both residual salt and ethanol are lethal to a developing embryo.

4. Bring up in a small volume of Injection Buffer (10mM Tris, pH7.4; 0.25mM EDTA - Please come get aliquot of injection buffer from the TPS) and determine the final concentration of DNA. The more concentrated, the better. Please provide samples at 50-100ng/µl (about 25-50µl). We will dilute the sample to the appropriate concentration for injection (usually 2 - 5ng/µl, depending on the size of the fragment) in Injection Buffer.

This protocol works well for us. Other facilities report using other protocols (e.g., centrifugation on CsCl gradients followed by extensive dialysis; or for large fragments electroelution followed by purification using the GeneClean Turbo Kit) with great success. If you choose to use an alternative method please provide us the protocol when you submit your sample. The DNA should be as clean as possible, free of salt and other contaminants that could result in decreased embryo survival, and prepared using a method that will not shear the fragment.