Services & Pricing
The services required vary for each project. Prices will vary from project to project based on the number of procedures required to accomplish each project. Please consult with the core manager for updated prices and individual project concerns.
ES Cell Culture Services
Non-IDDRC/CH members and non-IDDRC/non-CH members are charged 10% and 20% more respectively.
Standard transfection into either mouse J1 (129J/v) or Bruce4 (C57BL/6) or JM8 (C57BL/6) ES cells. We attempt to pick 192 colonies. There is no guarantee that clones that are positive for homologous recombination will be generated.
Cre transfection results in the picking of up to 48 clones and duplicate plating in G418 or without. Only clones that die in G418 are saved for screening. Usually 20% of Cre transfected clones are positive for recombination and therefore saved. Flp transfection results in less picked clones. For both Cre and Flp transfections, there is no guarantee that properly recombined clones will be generated.
More Details about ES Cell Culture Services
We currently use J1 cells, a 129 line. We do have a C57 line as well, but we should discuss that if you want C57. If all goes well with the transfection, we expect to pick 192 clones and freeze these in 4 96 well plates at -80. For secondary Cre or Flp transfections, we pick 96 clones. About 3 weeks following the transfection, we give you 24 well plates of cells for you to lyse to make DNA and genotype. You should get enough DNA for at least one Southern and often two. PCR genotyping assays are the most convenient for this stage of genotyping, but these need to be confirmed by Southern eventually. Once you identify positive clones, we thaw up to 24 positives and karyotype up to 8 of them. We grow more cells for you to make more DNA for further genotyping at this point.
We strongly recommend that you give us the genotyping results within 4 months of the transfection. Longer storage at -80 decreases the success rate of thawing the clones.
When secondary Cre or Flp transfections are planned, we advise injection of the first round homologous recombinant lines before transfection as well, followed by crossing the resulting mouse lines to Cre or Flp expressing. This approach has proved very successful and these lines are used as backups in case the in vitro recombined lines fail to transmit germline. While we routinely do Cre transfections with excellent results, Flp is less efficient in vitro.
Repetitive DNA warning: Reduce (eliminate if possible) repetitive DNA sequences in the homologous arms of the targeting construct. Repetitive DNA greatly reduces the targeting efficiency.
We request that people demonstrate that their genotyping assay for screening for homologous recombinants works on wild type ES cell DNA before having us do the transfection.
This is important because some probes surprise people with ugly smears indicating the presence of repetitive DNA in the fragment used for the probe. In some cases people have had to redesign their targeting vector to accommodate for a shifted probe fragment.
The requirements for proper genotyping assays are:
· PCR and/or Southern confirmation of homologous recombination on both 5’ and 3’ ends of the targeting construct.
· Southern probes that are external to the sequences used for the homologous arms of the targeting construct: i.e. probes must not be in the targeting construct.
· An independent verifiable assay for any mutations remote from the drug selection cassette.
· For PCR assays, one primer must be external to the homologous sequences in the targeting vector.
· PCR assays must be demonstrated to work on extremely low copy number in the presence of genomic DNA.
· PCR assays require the generation of a positive control plasmid that contains the external primer site as well as the change in the targeting vector. This control should be engineered to give a band of a size that differs from the ultimate homologous recombinant band to avoid cross- contamination confusion. An alternative approach to making a positive control plasmid involves designing primers that span the neomycin gene- ie. one external primer and one internal primer in the opposite homologous arm sequence. Demonstration that these primers work on wildtype DNA does not guarantee that they will work on the longer homologous recombinant DNA, however).
Please see “Genotyping Requirements” on the Policies page, point #3)
Please contact the Core to discuss genotyping further.
Before the transfection, we need to know
What are the positive and negative selections you need?
Will you want a secondary transfection with either Cre or Flp?
· Linearize 100 ug of DNA.
· Prove that the linearization worked by running a small sample of the digest on a gel without ethidium bromide, next to a lane of uncut targeting vector.
Purify the digest by:
· Phenol/Chloroform extraction,
· Chloroform extraction (very important to get rid of the phenol),
· Sodium acetate/ethanol precipitation.
· Bring us the DNA in the sodium acetate/ethanol. We will resuspend and read OD…we transfect 50 ug.
Mouse Embryo Microinjection
Non-IDDRC/CH members and non-IDDRC/non-CH members are charged 10% and 20% more respectively
Charges are per clone and will be charged in cases where the core transfers at least one high chimeric mouse (male or female) to the investigator. High chimeric means that the coat color of the chimera is at least 70% agouti (the color conferred by the injected ES cells). This is a subjective assessment by the core personnel. In many cases, one chimera is all that is needed to establish the desired line. In many, but not all cases, the core will give the investigator more than one chimera. While the core has a high success rate in generating chimeras that pass the desired mutations germline, there are no guarantees that germline transmission will be achieved. However, if for each chimera of > or = 70% coat color chimerism, after mating to generate 6 litters of 5 or more pups from each chimera, no F1 pups with the mutation are born, the core will inject the line again at least one time at no charge.
For pronuclear injections, price is for 2 F0 pups that genotype positive for the desired transgene. If the core injects 4 times, and no genotype positive pups are generated, the core will ask for a new DNA prep which it will try for a maximum of 2 more injections. If still no positive F0 pups are generated, the core may terminate the project and the charge will be $1000 for the effort. If the investigator chooses to have the core continue its attempts, the price will be determined on an individual project basis. If the investigator obtains 2 positive F0 pups and wishes the core to attempt to generate more, the price will be determined on an individual project basis. There are no guarantees that the mutation identified in an F0 pups will be transmitted to the next generation or that the transgene is expressed.
There is no guarantee that transgenic F0 pups will transmit the transgene through the germline.
Microinjection of ES cell lines into C57BL/6 or C57 Albino blastocysts and surgical implantation of injected blastocysts into pseudopregnant CD1 (or ICR) foster female mice.
The procedures required for making genetically altered mice are approved in the ARCH animal protocol; 11-04-1939R and the Harvard Committee on Microbiological Safety (COMS) license 01-105. However, the individual lines of mice generated by the IDDRC facility must be registered by the investigator with the Harvard COMS and approved in an animal protocol reviewed by the investigator’s institution’s animal care and use committee (IACUC).
Users need to arrange for the transfer of litters with chimeras during the week of postnatal day 7. ARCH will not approve transfer without proof that the animals are on the investigator’s animal protocol. Therefore animal protocol approval of the line is required before injection. If the animals are transferred after the week of P7, animal housing charges will be passed on to the user at twice the current ARCH housing rate.
For injection of “Outside” ES cell lines obtained from a source other than our Gene Manipulation Core:
-All cell lines need to be pathogen tested. Please use the Impact profile IV at the University of Missouri. http://www.radil.missouri.edu, or a similar service. Testing of “sister” lines that have been cultured in parallel to the injection lines is sufficient. (Most sources of ES cells have documentation of pathogen testing already)
-We strongly advise that all cell lines be karyotyped. Cell lines with good karyotypes may be given scheduling priority. The quality of the karyotype procedure and assessment must be confirmed by a member of the facility. A protocol for karyotyping is available upon request.
-For any given injection date, please plan on growing your cells according to our protocol (Prep of injection cells v.doc See Links and Resources) and bring us the single cell suspension by Noon on the scheduled injection date (usually a Friday). We can know on the Tuesday preceding the Friday injection how good a chance we have of being able to do two lines. Even if we feel we can only do one line, you may want to prepare multiple lines so that we can choose the line that looks the best.
When using J1 cells that were targeted in the IDDRC facility and that have high karyotypes (90 –100%), a single round of injection frequently (90% of injections) results in the birth of pups with a high percentage of agouti coat color indicating good chimerism. About 90% of these lines will “go germ line.”
Cells that are unkaryotyped and/or produced in facilities other than the IDDRC have been more variable.
Microinjection of DNA (traditional transgenes or BACs) into FVB/N (C57BL/6 or 129SvEv upon request) single cell embryos, and surgical implantation of injected embryos into pseudopregnant CD1 (or ICR) foster female mice.
- For the pronuclear injection of traditional transgenes, the core requires at least 0.25 ug of transgene purified away from plasmid backbone in at least 40 ng/ul concentration, along with a picture of a gel documenting the concentration estimate. (We calculate the amount of DNA to inject based on the documented concentration.)
-A currently successful DNA preparation is to Qiagen maxi-prep the plasmid. Digest and gel purify the proper fragment using the Qiagen gel extraction kit. After completion of the gel extraction kit directions, perform three rounds of EtOH/NaOAc precipitation and resuspend in TE that is 10mM Tris 7.5/0.25mM EDTA. Starting with about 10-20 ug of DNA, one can usually end up resuspending in a final volume of 12 ul or so to get in the right concentration range (this will of course vary with the construct, etc.) Estimate concentration using the Gibco/BRL Low mass ladder.
-Please contact Tam about details regarding the injection of BAC DNA.
- Users need to arrange for the transfer of litters with F0 pups during the week of postnatal day 7. The animals need to be transferred to the investigator’s animal housing and protocol. An approved animal protocol for the transferred animals is therefore required for the injection. If the animals are transferred after the week of P7, animal housing charges will be passed on to the user at twice the current ARCH housing rate.
We generally do not do tail biopsies because we export the animals before the time to biopsy. For the occasional times that animals stay longer, we will biopsy on a limited basis at no charge. In this case, we request that the investigator pick up the tails in CLS 12122.
We strive to do the best possible injection for you on the indicated date. However, the nature of the procedure is such that failures can occur at any of a number of stages, including, but not limited to a poor host embryo harvest on the day of injection, failure of pregnancies for known and unknown reasons, poor pup survival, low to no transgenic F0 offspring, no germline transmission of transgene, no expression of
transgene and pathogen contamination. We cannot guarantee successful pregnancies, pup survival, transgenics, germ line transmission, or that the animals will be pathogen-free.
Prices subject to change