Pricing and Services

Methods of pronuclear microinjection are patented under United States Patent No. 4.873,191, entitled "Genetic Transformation of Zygotes", assigned to OhioUniversity, and licensed to Xenogen. Roswell Park Cancer Institute has signed a sublicense with Xenogen that allows us to provide DNA microinjection services to any non-profit institution in the US. If you are a for-profit agency, you will need to obtain your own sublicensing agreement with Xenogen, naming Roswell Park Cancer Institute as the provider for your service.

Step 1: Perform TEST injection

Before beginning a full scale transgenic microinjection project for investigators, the GT/TG will first perform a TEST microinjection day. The investigator must supply a photo of the gel used to purify the DNA for microinjection to document its concentration and purity. In this test, the GT/TG will superovulate 5 F1 (B10 X C3H) females, harvest fertilized oocytes, inject your DNA into the pronuclei of these fertilized oocytes. The following day, we will assess the toxicity of your DNA to the embryos. If after microinjection, less than 50% of the oocytes injected develop to a 2-cell stage, the facility will ask the investigator’s lab to prepare the construct again; alternatively, the GT/TG facility can do this for you on a fee for service basis.

Death of the oocytes may be due to a range of contaminants, and thus is essential for the investigator’s lab to perform 2 CsCl gradients on each construct. Each time we receive a new “batch” of DNA, another TEST will be done. Surviving eggs will be implanted in pseudopregnant females.

Step 2: Production of transgenic mice by DNA injection of fertilized eggs

**Note: although the generation of founder mice is guaranteed, the expression of your gene is not guaranteed**

PER DAY (applicable to RPCI and SUNYAB investigators only):

The Gene Targeting and Transgenic facility will harvest and inject the pronuclei of F1 (B10 X C3H) fertilized oocytes with DNA prepared and purified by the investigator or the GT/TG facility (whichever is chosen). Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA, and will also be identified by an ear tag. The GT/TG guarantees that at least 100 eggs will be implanted or 3 transgenic offspring will be produced (whichever comes first). Surcharge for inbred donors for all RPCI and SUNYAB clients: $300 for C57BL/6J. The cost of your project will not exceed the cost of outside academic or industry.

PER PROJECT (applicable to outside academic and industry):

The Gene Targeting and Transgenic facility will harvest and inject the pronuclei of F1 (B10 X C3H) fertilized oocytes with DNA prepared and purified by the investigator or the GT/TG facility (whichever is chosen). Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA, and will also be identified by an ear tag. The GT/TG guarantees that 50 pups will be born or 3 transgenic offspring will be produced (whichever comes first). After weaning, investigators will be responsible for daily cage charges (see above).

Gene Targeting microinjection day(per cell line):

**Note: although the generation of chimeric mice is guaranteed, germline transmission is not guaranteed.  However, we have had a high percentage of our chimeras go germline (See: chimera generation report) **  As of 7/05: 57% of our mated chimeras have gone germline.

PER DAY (applicable to RPCI and SUNYAB investigators only):

The Gene Targeting and Transgenic facility will harvest blastocysts from C57BL/6 mice (currently C57BL/6J, from Jackson Labs) and inject 10-15 ES cells/blastocyst (either provided by the investigator or generated in our facility. If NOT generated in our facility, you must submit a Mouse Embryonic Stem Cell Pathology/Mycoplasm Report with your cell line) into each viable blastocyst. Injected blastocysts will then be implanted into pseudopregnant females. The GT/GT guarantees that at least 20 blastocysts will be successfully injected and implanted into foster moms, or 3 chimeric pups will be produced, whichever comes first.  The cost of your project will not exceed the cost of outside academic or industry.

PER PROJECT (applicable to outside academic and industry):

The Gene Targeting and Transgenic facility will harvest blastocysts from C57BL/6 mice (currently C57BL/6J, from Jackson Labs) and inject 10-15 ES cells/blastocyst (either provided by the investigator or generated in our facility. If NOT generated in our facility, you must submit a Mouse Embryonic Stem Cell Pathology/Mycoplasm Report with your cell line) into each viable blastocyst. Injected blastocysts will then be implanted into pseudopregnant females. The GT/GT guarantees that 50 pups will be born or 3 chimeric pups will be produced, whichever comes first.

Electroporation:

1) Gene Targeting:

Upon receiving your lab’s DNA construct, the Gene Targeting and Transgenic facility will perform an electroporation of your DNA into 129SVJae, C57BL/6J or Balb/c ES cells. Colonies will be selected with the appropriate drug, depending on your positive selectable marker. Colonies can also be negatively selected for enrichment purposes. Selection methods should be discussed with the facility at least one month prior to the electroporation date to prepare the necessary feeder cells. The facility will pick and freeze ~240 colonies of which we will give the investigator’s lab ~200 DNA preps. The investigator will screen these clones by Southern analysis, and notify the facility of the positives for homologous recombination. The facility will expand 5 of these clones and give the investigator DNA again to reconfirm positives. When reconfirmed, the facility can begin Gene Targeting microinjections for the investigators laboratory. The entire electroporation process takes approximately one month to complete and the facility expects the investigator’s lab to perform the positive confirmations in a timely fashion.

2) ES cell-based transgenics

We have had multiple requests for, and have successfully performed ES cell-based transgenesis in the past. ES cell-mediated random insertional transgenesis is not only an alternative for producing transgenic mice, but it also has its own advantage when there is a need for low copy number (single copy) integration. In addition, it also allows checking of expression of the transgene before a decision is made to introduce the transgenic cells in vivo. Please refer to the third edition of Manipulating the Mouse Embryo by Nagy et al. for vector construction information. Because it is an integration event, and does not rely on homologous recombination, the frequency is much higher (90-99%) than a classical electroporation (0.1-10%). This is the reason why the cost is reduced: The Gene Targeting and Transgenic facility will perform the electroporation as described above, but we will only need to pick 50 colonies/electroporation, as opposed to 240. This will result in a reduction of supplies used, and thus reduce our cost. ES cell-based transgenesis also allows for generating mice on backgrounds which pronuclear injection is very difficult to impossible. Also, the DNA used does not need to be isogenic to the ES cell strain as it does with traditional gene targeting.