Transgenic Core, Rederivation, Cryopreservation and Cryo-Recovery Services

• Leica Labovert FS microscope with dual micromanipulators
• Sutter Flaming/Brown Micropipette Puller P-87
• Narashige microforge MF-9
• Olympus SZH10 dissecting stereoscope
• Eppendorf Transinjector 5246
• Laminar flow hood
• Nuaire CO2 incubator Model NU 5500 series 12

Generation of Transgenic Mice and CRISPR/Cas 9 modified Mice

Transgenic mice are generated by the microinjection of purified DNA into the pronuclei of one-cell fertilized mouse embryos. CRISPR/Cas9 are produced with similar microinjection into the cytoplasm and pronuclei to produce targeted genetically-modified mice.

Generation of Knock-out Knock-in Mice

The Transgenic core microinjects ES cells into mouse blastocysts for investigators who have either generated targeted clones in their lab or obtained ES cell clones from another source.

Rederivation

Rederivation is used to eliminate undesired pathogens from infected mouse lines. Rederivation allows the import of established mouse lines with either known pathogens or of unknown health status.

Sperm and Embryo Cryopreservation

Cryopreservation is the process of freezing germplasm for stable, long-term storage in liquid nitrogen. It is a safe method for preserving viable sperm or embryos that can be easily recovered at a future date to revive the line.

Additional Services

Our cryo-recovery service allows the worldwide import and recovery of mouse lines from frozen embryos and sperm. The core also provides mouse colony management for a limited number of investigators. Additionally, the Transgenic Core Facility provides mouse breeding advice to any College of Medicine investigator.

DNA must be linearized and purified by the approved DNA Preparation Protocol. Investigators who wish to use another protocol must first get approval from Transgenic Core personnel.

DNA purity and concentration of the injected DNA is one of the most critical factors for the success of transgene injections. It is the investigator’s responsibility to provide high-quality DNA that has been linearized and digested to remove all bacterial vector sequences. Prokaryotic sequences have been observed to inhibit transgene expression in a number of cases. Particles within the DNA could easily clog the microinjection needle and interfere with integration of the DNA.

Conditions for transgenic identification with proper controls should be worked out well in advance to avoid delays in identification of founders.

All users of the Transgenic Core Facility must demonstrate competency in DNA preparation and analysis from tail clips. DNA probes and restriction enzymes should be chosen to differentiate between plasmid contamination and true positives (i.e., two different enzymes should be used that cut only once or do not cut the transgenic construct at all.)

The investigator must supply a photo of a EtBr-stained gel (with markers of known concentration) and have the DNA analyzed by the Functional Genomics Core to document DNA concentration and purity. Contact the Functional Genomics Core in Room C3603, 717-531-5823, or email Rob Brucklacher at rbrucklacher@psu.edu for more information. A copy of the analysis and a brief description of the construct and its expected phenotype are requested.

The DNA should be at a concentration of 4ng/ul with a volume of at least 150 ul and suspended in sterile MiTE (microinjection buffer): 10mM Tris-HCl, pH 7.4, 0.1mM EDTA (higher concentrations of EDTA are toxic to embryos.)

DNA must be delivered to Room ARF 173 no later than noon of the Friday before the first injection is scheduled. DNA should be labeled for easy identification. Investigators must arrange a dropoff time to ensure the lab is unlocked or someone is available.

All required paperwork must be sent to Alane Seidel at MC HS65, emailed to aks2@psu.edu, faxed to 717-531-4715 or left on the desk in the office of ARF 173 or Alane Seidel’s mailbox in the ARF office.

The DNA submitted to the Transgenic Core is immediately divided into three separate tubes, with some of the sample left in the original tube so it can be checked if a problem arises with the DNA. Two of the tubes are used for the standard two injections and the third is kept in the event that a reinjection is needed. These tubes are saved and can be used to check the quality of the DNA after storage, dilution and injection if a problem arises.

To perform DNA purification for transgene microinjection:

• Recombinant plasmid should be purified by CsCl gradient or Qiagen Maxi-prep. The insert must then be separated from the vector by restriction digestion, since vector sequences can significantly alter the expression of transgenes.
• Separate the insert from the vector on an agarose gel run in Tris/Acetate/EDTA (not Tris/Borate/EDTA) buffer. Use 5 mg/ml Ethidium bromide for gel staining. Visualize the DNA with long wavelength UV light to avoid damaging the ethidium bromide-intercalated DNA.
• Excise the gel slice containing the gene fragment of interest and electroelute the DNA, or else process though Qiaex gel extraction kit (Qiagen Inc.) according to manufacturer’s instructions.
• Ethanol precipitate the DNA. For ethanol precipitation of sample, add 1/10 volume of 3 M NaAcetate, mix, then add 2 to 2.5 volumes of 100 percent ethanol. Incubate at -20 degrees C overnight, then spin 5 minutes in a microcentrifuge to pellet the DNA. Resuspend in Elutip buffer (Schleicher and Schuell).
• Pass the DNA through an DEAE Elutip-D mini-column. (Schleicher and Schuell).
• Ethanol-precipitate the DNA as above. Wash the pellet several times with 70 percent ethanol and dry the pellet under vacuum. The washing and drying steps are very important, as residual salt and ethanol are lethal to the developing embryo. Resuspend in sterile MiTE (10mM Tris/0.1mM EDTA, pH 7.4 prepared with Sigma embryo-tested water or Milli-Q quality water.
• Estimate the DNA concentration. Run a small sample of the prepared fragment on an agarose gel alongside uncut plasmid and a set of known standards such as lambda-HindIII markers. A photograph of this gel must accompany all requests for microinjection. Adjust the DNA concentration to around 4ng/ul in MiTE and make sure there is at least 150 ul of volume to be submitted to the Transgenic Core Facility.

Tubes must be labeled with the concentration and volume and the name for the DNA construct before submitting the DNA to the Transgenic Core.

The DNA should be brought to Room C1732 (Macromolecular Core Facility) no later than noon of the Friday before the first injection is scheduled. The DNA should be put that room’s second freezer on the left, just past the second door on the left, and should be properly labeled for easy identification.

All paperwork required should be placed in the tray inside the door of Room C1735 (Mass Spectrometry/Proteomics Core), not left in Room C1732.

• Charles River Laboratories
• Envigo Scientific Insights
• JAX Mice and Services
• Mouse Genome Informatics (a JAX service)
• Mutant Mouse Resource and Research Centers
• Taconic

Investigators should cite Penn State College of Medicine Transgenic Core and list the names of the instruments that were utilized either in the Materials and Methods section or the Acknowledgements section of any article.

Transgenic setup and injection fees apply for mouse strains B6D2F1, C57BL/6, FVB and investigator-provided strains.

When strains are provided by the investigator, male and female mice needed for production of fertilized embryos for injection must be supplied. These mice must be kept under the investigator’s mouse protocol and be proven pathogen-free before being moved into the transgenic mouse room. No guarantee applies to these mice.

For knock-out mice, fee schedules are available for one-injection days, three-injection days and five-injection days.

For rederivations, setup and rederivation fees apply, and service is available for one line.

For mouse sperm cryopreservation, three-strain kits, six-strain kits and nine-strain kits are available at the cost of the kit plus an hourly charge for staff time (normally about one hour per strain).

For fee details, email Alane Seidel at aks2@psu.edu.

All Transgenic Core procedures are scheduled on a first-come, first-served basis. Procedures that include ordering mice will be scheduled according to the arrival of the mice. In some instances, mice may be backordered and the procedures will be delayed accordingly. No procedures will be scheduled until the appropriate form is received by the Transgenic Core.

Any animal care or welfare concerns should be addressed to Dr. Danielle Covington, DVM, DACLAM, at dcovington1@pennstatehealth.psu.edu.