Instructions & Forms
Transgenic Animal Model

Transgene Sample Instructions
Targeting Vectors Instructions
Sample Preparations
Forms
Material Transfer Agreements

Transgene Sample Instructions

  1. We will purify the DNA for you. Simply perform a restriction enzyme digest on your cloning vector to liberate 50 ug of the transgene insert from the cloning vector.
  2. Run out a few hundred nanograms of DNA on a minigel to determine that the digest went to completion and that the bands are the correct size. Bring a photo of the minigel to submit with your sample. Mark the bands on the photo to show which band should be purified for microinjection.
  3. Bring the remainder of the digest (in a final volume of 100 to 150 microliters) to the Core lab and we will purify the DNA for microinjection from the digest. We use either the Gel Extraction Kit (Qiagen) or the Machery Nagel Nucleospin extract kit (Clontech) for purification of microinjection DNA. Please note, if you want use large DNA fragments such as bacterial artificial chromosomes, that there is a different protocol for the preparation of the BAC DNA for microinjection.
  4. Bring a gel photo that shows you have a PCR assay for an endogenous mouse gene such as beta-globin. All DNA samples from potentially transgenic mice should give a positive result with an endogenous gene PCR. This will make sure that no transgenic founders are discarded because PCR inhibitors co-purified with the genomic DNA.
  5. Bring a gel photo that shows your genotyping PCR. Test the PCR against 10, 1, 0.1 and 0.01 copy standards. The assay must detect the transgene at the single copy level, preferably 0.1 copies, when it is mixed with mouse tail DNA. Bring your calculations of copy number along with the gel photo. If you need tail DNA to set up the assay, we will give you some.
  6. Bring a submission form with your contact information and billing information.
  7. Drop off all your materials at the microinjection lab: Room 2526, Building MSRB I.

Transgene constructs are purified, quantitated, and microinjected into (C57BL/6 X SJL)F2 mouse eggs and surgically transferred to recipients. Other mouse eggs can be used for transgenic production when prior arrangements are made with the Core. Contact Thom Saunders regarding custom mouse strains.

Transgenic mouse production is a fee for service provided by the Transgenic Core. The Core prioritizes all requests for service on a "first-come, first-serve" basis. Your DNA will be added to the microinjection queue in the order that it is received.

Targeting Vector Instructions
Targeting vector DNA are used to modify genes in mouse embryonic stem cells.This process is complex, involving many procedures that are carried out over a year or more. Careful attention to detail in the design stage often makes the difference between a smooth, successful experience and a harrowing, successful experience. Investigators are invited to contact Thom Saunders for tips on planning an experiment.

  1. Prepare the targeting vector DNA as described.
  2. Fill out the electroporation submission form.
  3. Bring the linearized targeting vector DNA, the submission form, and backup materials (evidence of a screen to detect homologous recombination in 96-well plate ES cell DNA) to the Mouse Embryonic Stem Cell Laboratory (2578 MSRB II).

Sample Preparation
For the production of conventional transgenic mice, investigators are responsible for providing a restriction digest containing 50 ug of the transgene insert. The Transgenic Core will purify DNA for microinjection. A minigel photo should accompany the digest, the DNA fragment to be purified should be clearly marked. Several things should be considered in designing a transgenic research project (see Transgenic Project Outline). Prokaryotic vector sequences interfere with the expression of some transgenes, thus unique restriction sites at the 5' and 3' ends of the construct should be available for vector removal. The transgene should contain unique markers so that its presence can be easily detected in DNA samples and its expression can be assayed and distinguished from endogenous gene expression.

Certain transgenes may result in a very low yields of transgenic founders due to the intrinsic nature of the transgene. For example, certain genes will be deleterious or incompatible with proper growth and development of the embryo. Special arrangements should be made with the Core if the transgene is suspected to cause lethality. The expression of a transgene requires that the appropriate transcriptional control elements be included in the DNA construct. Expression is often influenced by the chromosomal site in which the transgene DNA is integrated. Preliminary studies in cell cultures are recommended to verify the integrity of the construct and the function of the promoter/enhancer. However, it is not always possible to predict in advance whether the transgene will be expressed in vivo. For these reasons, the Core cannot guarantee that transgenic founders will express the transgene.

For the production of gene targeted mice with mutations induced by homologous recombination in ES cells, investigators are responsible for producing ES cell clones with targeted genetic mutations. The first step in gene targeting is to obtain a detailed restriction map of the genetic locus and knowledge of exon/intron boundaries. (see Gene Targeting Outline) Higher yields of targeted ES cell clones are obtained from isoegenic DNA, thus a clone(s) from a genomic library that exactly matches the ES cell line to be electroporated is strongly advised. Restriction mapping data is used to identify 5' and 3' arms for targeting vector construction and to establish a screening strategy for the identification of ES clones that have undergone homologous recombination. This requires the characterization of probes for Southern blot analysis that lie outside of targeting vector sequences. After the targeting vector has been cloned it is introduced into ES cells. Fastidious cell culture technique and specialized reagents are required to maintain ES cells in a pluripotent state. Differentiated ES cells will not produce chimeras with the ability to transmit targeted mutations through germ cells. Investigators are invited to contact Elizabeth Hughes for training in ES cell culture and to obtain reagents certified for ES cell culture. Due to the intrinsic variability of individual ES cell clones we cannot guarantee that germline chimeras will be produced from any particular ES cell clone. The Transgenic Facilty tests and validates ES cell culture reagents to maximize the successful outcome of gene targeting projects.

Forms

129X1/SvJ Library Aliquot
BAC DNA Purification
BAC Recombineering
Blastocyst Injection Request
Cryo Recovery Request
Cryopreservation Request
Cryopreserved Mouse Shipping
De Novo ES Cell Derivation
ES Cell Electroporation
ES Cell Training
Expand Clone from Electroporation
In Vitro Fertilization Request
KOMP Clone Expansion
Mouse Transgenic Production
Rat Transgenic Production
Speed Cryo Request
Sperm Cryopreservation Request
SPF Rederivation Request