To produce transgenic mice, a purified transgene prepared by the investigator according to the provided protocols DNA Preparation Protocol (PDF) is microinjected into pronuclei of the mouse zygotes and implanted into pseudopregnant recipients. Pups are screened for the presence of a transgene at 2-3 weeks of age. Transgenic founders are identified by investigator and used for breeding to establish transgenic line.
Following backgrounds are currently available for pronuclear microinjections of zygotes: outbred ICR, inbred FVB/NJ as well as the embryos from the cross of C57BL/6 females with B6D2F1 (C57BL/6 x DBA/2) males.
Please, inquire about the cost and feasibility if other background strains are required for the transgenic production.
Clone and verify the transgene. Use unique restriction sites at the ends of the construct for vector removal as prokaryotic vector sequences often interfere with the expression. Unique markers such as reporter genes such as GFP, LacZ, non-mouse proteins or epitope tag are necessary to assay the expression and distinguish it from the endogenous gene. Sequence the junction fragments to confirm the presence of a functional promoter, initiation codon and polyadenylation signal. If at all possible, test the transgene for expression in a tissue culture system before attempting the generation of transgenic mice. Please, refer to the publications listed below for details.
Establish a screening method and provide the evidence that you have a PCR or Southern blot assay that detects the transgene when it is spiked into the tail DNA at 2. a single copy concentration. A second assay that will detecting an endogenous mouse gene (e.g. beta-globin) will can demonstrate that the DNA preparations are suitable for PCR. When animals are tested with both assays, no transgenic founder is mistakenly discarded as false negative. A Southern blot assay is needed to determine the copy number, integration site number, and transgene integrity in the transgenic founders prior to breeding. More information on the preparation of copy standards and genotyping sensitivity (see Protocol 1, Protocol 2) sis available courtesy of Dr. Thom Saunders, University of Michigan Transgenic Animal Model Core Facility -
Purify DNA fragment following the DNA Preparation Protocol (PDF) The quality of DNA preparation is the main factor affecting the efficiency of transgenic production. DNA should be pure of any contaminant and at the appropriate concentration. Perform a restriction enzyme digest on the cloning vector to separate 50 ug of the transgene insert from the cloning vector. Run out a few hundred nanograms on a minigel to determine that the digest is complete and that the bands are of the correct size.
Prepare 50-100 ng/ul in at least 50ul volume. It is strongly recommended to use NanoDrop to quantify and assess the purity of the final prep BEFORE submitting the service request. Please, provide NanoDdrop results to TG core staff, refer to NanoDrop Nucleic Acid Purity ratios (PDF)
Once DNA prep is ready, submit a service request: Login to LIMS and from the top menu go to Service Requests -> Request a TCP Service page
, search with Department set to "TCP Transgenic Core and Specialty Resources", then select appropriate service request for Pronuclear Microinjection
Provide the service request form and attach the following gel photos and purified DNA fragment at the required concentration and volume:
Assay detecting the transgene at a single copy level when it is mixed with tail DNA as well as any endogenous gene
Comparative gel analysis determining the accurate concentration of the prepared DNA fragment with known amounts by relative intensity of the bands run side by side
Bring the documents and purified DNA fragment of required concentration to TCP