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Pronuclear Microinjection

Service Details      Service Charges      How To Order


Service Details

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.

Guidelines:

  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. Please, refer to the publications listed below for details.
  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 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 -
  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, provide NanoDdrop results to TG core staff, 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.
  8. Advanced Protocols for Animal Transgenesis: An ISTT Manual. S.Pease, T.L.Saunders (eds) Springer 2011.

Service Charges

Service Mount Sinai &
Sick Kids
Hospitals
External Canadian Academic Other External Academic
Pronuclear microinjection into CD-1(ICR) zygotes (per attempt) $2182 $2836 $3491
Pronuclear microinjection into FVB/N zygotes (per attempt) $2291 $2977 $3664
Pronuclear microinjection into B6x B6D2F1 zygotes (per attempt) $2283 $2968 $3653
    The fees include:
  • purchase of embryo donor animals and their housing prior to the experiment (estimated based on the strain background, number of animals and the source)
  • one microinjection session - see service agreement
  • transfer of microinjected embryos into pseudopregnant recipients (estimated based on 8 recipients and overnight culture of microinjected embryos)
    The fees do not include (extra charges apply):
  • animal housing (per diem) charges for recipients and their offspring from the time of embryo transfer surgery
  • health monitoring (test of fosters, one per attempt)
  • tissue biopsy
  • weaning
  • 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. PI and the lab contact 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, provide the results to TG core staff including NanoDrop profile when available - 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 appropriate service request for Pronuclear Microinjection
  7. 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
    • NanoDrop results
  8. Bring the documents and purified DNA fragment of required concentration to TCP

 

 

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