Preparation of Transgene DNA  for Microinjection

 

 

The purity of the transgene DNA used for microinjection is critical for the successful production of transgenic founder mice. DNA impurities in the form of bacterial endotoxins or organic contaminants can decrease the integration efficiency of transgene DNA or the viability of microinjected embryos.

A transgene construct prepared from genomic DNA is preferential to one prepared from a cDNA clone. While there are some reports of cDNA transgene expression in the literature, introns in the DNA increase the likelihood of transgene expression. Plasmid vector sequences should also be cleaved away from the transgene construct to be used for microinjection. The presence of plasmid sequences interferes with transgene expression. Listed below are guidelines for the preparation of DNA for microinjection.

Use plasmid DNA that has been purified on a cesium chloride gradient to obtain DNA of highest purity. Digest approximately 20 µg of plasmid DNA with a restriction enzyme that will remove the plasmid sequences from the transgenic DNA.

 

Run the DNA on an agarose gel and elute the band containing the transgenic DNA to be injected. Most elution methods work fine. We have used GeneClean from Bio101. The QiaEx gel extraction kit from Qiagen will also work. Phenol free methods that leave little salt contamination are desirable.

 

Resolubilize the eluted DNA in injection buffer:

10 mM Tris, pH 7.5

0.1 mM EDTA, pH 8.0

When preparing the injection buffer, use disposable plastic, free of traces of detergents. Use reagents designated solely for tissue culture work to avoid trace contaminants. Solutions should be filtered with a 0.2 µm filter to remove particulates which may clog the injection needle. The purity of the DNA sample to be microinjected must be documented by digesting with at least 2 enzymes that cut within the transgene and produce fragments of the predicted sized.

Submit a gel to verify the purity and determine concentration of the DNA. Supply 1-5 µg of DNA adjusted to a final concentration of 100 ng/µl. We will dilute the DNA to 1-3 µg/ml for microinjection. Too high a DNA concentration will cause developmental arrest of embryos. Too low a concentration decreases the number of transgenic founders. DNA concentration is best determined by preparing serial dilutions of lambda DNA of known concentration (25, 50, 100, 200, 400 ng of lambda DNA) and using them as standards. Run the lambda DNAs and the DNA to be microinjected on a gel to determine the DNA concentration of the diluted DNA for microinjection. We will also verify the concentration by fluorimetry.