Preparation of DNA for Microinjection

Digest 100 micrograms of the transgene plasmid with restriction enzymes that will separate the vector from the transgene. Use Qiagen or CsCl-purified DNA for this digestion – sequencing-grade purity of DNA should be sufficient (i.e., unusual purification steps are not required). Digest the DNA in a volume of 100-200ml. DO NOT purify the transgenic fragment – the core facility will perform this purification. Provide the facility with the digested DNA along with an image of an Ethidium bromide-stained agarose gel showing which bands should be purified for microinjection.  BAC DNA should be provided to the core facility in supercoiled or linearized form at high concentration.  Core personnel will dilute the BAC DNA with an appropriate buffer before microinjection.

Note carefully: By far the most robust way to screen initially for transgenic founders is to perform a Southern blot using DNA prepared from tail biopsies of mice. Southern blots are preferable to PCR-based assays at this stage because they unequivocally identify founders and are much less prone to false-negative or false-positive results than other assays. When designed appropriately, the Southern blot can readily provide information about transgene copy number. It can also help to make clear when a founder is a mosaic (with only some of its cells carrying the transgene) or when a founder carries independent chromosomal integrations of the transgene (in such instances, more than one transgenic line can be produced by mating the founder and these lines may differ in expression characteristics). Screens for founders based on transgene expression can accompany Southern blot assays, but ideally should not replace them. Accurate information concerning the success of a microinjection experiment can best be obtained from a Southern blot.

Following the identification of founders (and ideally also their F1 progeny) PCR assays can be established for routine screening for transgenic mice. Ideally, such assays should include internal control reactions. Quantitative real-time PCR assays can also be developed to distinguish heterozygous from homozygous transgenic mice.

The core facility will take every opportunity to keep investigators informed concerning progress on their microinjection experiments. In turn, the facility depends heavily on the investigators for accurate feedback concerning transgenic founder frequencies and germline transmission from ES cell-derived chimeric mice.