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Pronuclear Microinjection Services
The purity of the microinjected DNA fragment is critical for successful production of transgenic founder mice. DNA impurities in the form of bacterial endotoxins or organic contaminants can decrease integration efficiency of the fragment or the viability of the microinjected embryos. For this reason the TCF will purify the DNA fragment for microinjection from plasmid DNA provided by the investigator.
- The following conditions must be met by the investigator for submission of plasmid DNA:
- A map of the plasmid, delineating the insert and relevant restriction sites.
- Plasmid DNA is purified preferably on a cesium chloride gradient,
or using of the Qiagen Plasmid Maxi kit.
- The presence of prokaryotic sequences interferes with transgene expression. Thus the insert to be injected must be flanked by restriction site(s) that allow removal of vector sequences. The purity of the DNA preparation is to be documented with a gel photograph. The plasmid should be digested with the enzymes(s) used to release the insert from vector sequences, as well as two enzymes that cut within the insert and produce fragments of predicted size.
- A genotyping strategy with sufficient sensitivity to detect 0.5x copy of the transgene must be demonstrated.
The Facility also notes that a transgene construct prepared from genomic DNA is preferable to one prepared from cDNA. Although there are reports of cDNA expression in the literature, the presence of introns increases the likelihood of transgene expression.
Once all of the above criteria are met, please submit a completed electronic application, DNA construct, and supporting documentation. Users will be scheduled for microinjection services on a first-come, first-serve basis. Notification of the scheduled microinjection date will be sent via e-mail within 10 working days of the receipt of all application materials.
The application and supporting documentation can be faxed to 617-636-8362, or hand delivered to the facility located on the 1st floor of the Tupper Research Institute at 15 Kneeland Street. Once the application is approved, the investigator will provide 100 micrograms of high purity plasmid DNA (as described above) to the facility.
Following purification and quantification of the DNA fragment, the construct will be microinjected into one-cell mouse embryos. The TCF produces transgenic mice on an B6D2F1 X B6D2F1 genetic background. Alternate backgrounds can be used, but Investigators should discuss these preferences with the TCF prior to submitting an application, as prices may vary.
The TCF earpunches mice at postnatal day 21 and provides tail samples to the investigator. The investigator is responsible for genotyping potential founder transgenic mice and establishing the line of mice from each founder. The TCF strongly recommends screening potential founders by Southern Blot.
Cost
Upon initiation of the project, a 50% non-refundable deposit is due. Upon completion of the project, the remaining 50% of the fee is due. If there is no founder animal carrying all of part of the transgene, the remaining 50% fee will not be assessed.
Tufts Medical Center Investigators: $2,650 *
Tufts University Investigators: $4,000 *
External Academic Non-Profit Investigators: $4,000 **
Industry: Contact the Administrator for pricing.
*Price excludes the purchase of female donors and per diem expenses, which are charged directly to the investigator by DLAM. Contact the Core for an estimate of the additional charge.
**Price excludes the purchase of female donors and per diem expenses. The actual amount incurred by the TCF for these purchases will be added to the final bill. Contact the Core for an estimate of the additional charge.
Note, the following groups are eligible for a cost subsidy:
MCRI Investigators
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