Pronuclear Microinjection

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Service Details

To produce transgenic mice, a purified transgene 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 microinjections: outbred ICR and inbred FVB/N as well as 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.

Guidelines:

1. 1.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.
2. 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 a sigle copy concentration. A second assay that will detect an endogenous mouse gene (e.g. beta-globin) 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 -
3. 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.
4. 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, refer to NanoDrop Nucleic Acid Purity ratios (PDF)
5. For the large DNA fragments such as bacterial artificial chromosomes, use the protocol BAC DNA Purification for Transgenic Mouse Production

References

1. Methods in Enzymology. Guide to Techniques in Mouse Development. 1993. Edited by Paul M. Wassarman and Melvin L. DePamphilis. Vol. 225.
2. Manipulating the Mouse Embryo: A Laboratory Manual. Hogan B, Beddington R, Constantini F, Lacy E. 1994. Cold Spring Harbor Press. New York.
3. Transgenic Animal Technology: A Laboratory Handbook. 2nd Edition. Pinkert, CA. 2002. Academic Press, New York.
4. Transgenic Mouse Methods and Protocols. Methods in Molecular Biology vol. 209. Hofker, MH,and van Deursen, J. 2002. Humana Press. Totowa, New Jersey.
5. Animal Transgenesis and Cloning. Houdebine, L-M. 2003. Wiley, John & Sons, Incorporated. Hoboken, New Jersey.
6. Manipulating the Mouse Embryo: A Laboratory Manual. Nagy, A, Gertsenstein, M, Vintersten, K, Behringer, R. 2003. Cold Spring Harbor Press. New York.
7. Mammalian and Avian Transgenesis: New Approaches. Pease, S and Lois, C. 2005. Springer-Verlag. Berlin, Germany.

Service Charges

Service	Mount Sinai and Sick Kids Hospitals and University of Toronto Faculty of Medicine	External Canadian Academic	Other External Academic	For Profit Organizations
Pronuclear microinjection into ICR zygotes (per attempt)	$2035	$2646	$3256	$4070
Pronuclear microinjection into FVB and B6 x B6D2 zygotes (per attempt)	$2195	$2854	$3512	$4390
Service fees include: purchase of embryo donor animals and their housing prior to the experiment one microinjection experiment implantation of microinjected embryos into pseudopregnant recipients tissue biopsy at 2-3 weeks of age (ear/tail tissue collection) for genotyping by the investigator Service fees do not include: animal housing (per diem) charges for recipients and their offspring from the time of embryo transfer surgery repeated tissue biopsies export fees and shipping charges

How to Order

1. Refer to Policies and Procedures for the required documentation prior to the initiation of the experiment and Service Agreements
2. Follow the guidelines above about the transgenic construct
3. Register for TCP LIMS account
4. Prepare DNA as described above and purify it following the DNA Preparation Protocol (PDF).
5. Quantify and assess the purity of the final prep - refer to NanoDrop Nucleic Acid Purity ratios (PDF)
6. 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 the Pronuclear Microinjection service request
7. Print the service request form and attach the following gel photos:
   
   • 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
   • NanoDrop print out
8. Bring the documents and purified DNA fragment of required concentration to TCP