Pricing

Core Description

This core facility is designed to facilitate the creation of new mutant mouse strains for diabetes-related research. The main emphasis of the core is to provide diabetes investigators access to services that are not readily available from commercial entities and that would require prohibitive investment of time and effort for them to establish in their own laboratories. Thus, the core provides expertise in the areas of gene-targeting in mouse embryonic stem (ES) cells and in microinjection of mouse embryos. The core is focused on the importation and use of cell lines, reagents and other resources that will facilitate investigations into the mechanisms that cause diabetes.

Services Provided

·          Microinjection of DNA into fertilized mouse oocytes to create transgenic mice

·          Microinjection of ES cells into mouse blastocysts to create chimeric mice

·          Gene targeting in ES cells

·          Culture and transfection of ES cells

·          Selection, isolation and freezing of clones of ES cells

·          Preparation of DNA from clones of ES cells

·          Digestion of DNA and preparation of Southern blots for screening cloned ES cells

·          Expansion of targeted ES cells from 96-well plates

·          Rederivation of mouse strains by embryo transfer

Pricing

·          Primary gene targeting experiment (129/Sv ES cells)                          $2000

·          Expansion of individual clones of ES cells                                            $400

·          Secondary transfection of gene targeted clones (e.g. with cre)                      $1000

·          Microinjection of targeted ES cells                                                       $2000

·          Microinjection of DNA into FVB/N oocytes                                             $2000

·          Microinjection of DNA into B6D2 oocytes                                              $2000

·          Microinjection of DNA into NOD oocytes                                               $2500

·          Microinjection of DNA into C57BL/6 embryos                                        $4000

·          Rederivation of mouse strains by embryo transfer                              $650

·          Primary gene targeting experiment (C57BL/6 ES cells)                                    $2400

·          Primary gene targeting experiment (NOD or other ES cells)                $2600

·          Microinjection of targeted C57BL/6 ES cells                                         $2400

Placing an Order

DERC investigators should send a brief email message to Stephanie Louie requesting instructions on how to place orders for services

Contact Information

Nigel Killeen     Core director                502-5495         HSE1001E
Mark Anderson Assoc. director             502-8052         HSW1112
Ayako Kuroda                                      502-1333         HSE1001
Li Lu                                                     502-5432         HSE1001

Transgenic Mice

Two or more days of microinjection are required to complete the injection of up to 200 embryos with DNA. The injected embryos will be transferred to pseudo-pregnant recipient mice.  Implanted dams will be maintained in the facility until their pups can be weaned and delivered to the requesting investigator.  10-30% of the transferred embryos give rise to viable mice, and of these typically 20% carry the transgenic DNA in their germlines (i.e., about 5-10 transgenic founders should be expected).  The efficiency of generating transgenic mice using NOD, C57BL/6 mice or most inbred strains is lower than when using FVB/N or hybrid mice, thus the costs are higher for these experiments.  Investigators with special needs are encouraged to contact the core to discuss them.  Details concerning the preparation of DNA for microinjection can be found here.

Gene Targeting

Embryonic stem (ES) cells will be electroporated with targeting vectors provided by investigators. Instructions for the preparation of targeting vectors for transfection can be found here.  Transfected cells will be plated and subjected to drug selection. Clones of cells will be picked and replica-plated in 96-well format. One replica plate of cells will be frozen when confluent; DNA will be prepared from the other plate and given to the investigator for analysis. Each gene targeting experiment will provide the investigator with DNA from 100-500 clones of cells for analysis, depending on the nature of the construct, drug selection strategy and the efficiency with which clones are generated. The DNA purification procedure employed by the core is best suited to analysis by Southern blot, but can also be used for PCR screens.

The core facility has experience using a variety of ES cells for gene targeting experiments. The preferred choice at present is the E14Tg2A.4 feeder cell-independent line from 129/Ola mice prepared by Dr. William Skarnes (The Wellcome Trust Sanger Institute, Cambridge, England).  Other ES cells (e.g., C57BL/6 ES cells) are also available, but the costs associated with using them are higher.

ES cell clones identified as interesting by an investigator will be thawed, expanded and refrozen.  DNA will also be extracted from the thawed clones, and this will be provided to the investigator for confirmatory analysis prior to microinjection.  Up to 12 clones will be thawed and expanded for the indicated cost.  Note that the thawed plates will not be refrozen, so clones can only be recovered from a given plate at one time.

Gene-targeted ES cells can be retransfected with Cre or Flp recombinase expression vectors as required by the investigator.  The cells will be replated and subjected to drug selection if necessary.  Clones of cells will be picked, expanded and frozen down.  96-well plates of DNA will be returned to the investigator for analysis.

1-2 days of microinjection are usually required to complete the injection of up to 100 blastocysts with 2-3 clones of ES cells. The injected embryos will be transferred to pseudo-pregnant recipient mice. Implanted dams will be maintained in the facility until their pups can be weaned and delivered to the relevant investigators. 30-50 injected embryos are usually sufficient to generate chimeric mice that are representative of what can be expected for a given ES cell clone. Details concerning the preparation of ES cell suspensions to be used for microinjection are provided here. The core facility will microinject ES cells from the gene targeting experiments it performs; it will also microinject ES cells provided by investigators.

Rederivation of Mouse Strains

The core personnel can perform embryo transfers to facilitate the introduction of mouse strains into the Barrier Facility.  Personnel will recover embryos from timed matings set up by the investigator.  The embryos will be transferred at the one-cell or blastocyst stage to pseudo-pregnant recipients maintained within the Barrier Facility.  Pups from the implanted dams will be delivered to the investigators after weaning.  DERC investigators are advised to make use of a LARC core facility that has been established specifically for this purpose.

Consultation

Advice about the design of transgenic or gene targeting experiments can be obtained from the Core Director.

Preparation of DNA for Microinjection

Digest 100 micrograms of the transgene plasmid with restriction enzymes that will separate the vector from the transgene. Use Qiagen or CsCl-purified DNA for this digestion – sequencing-grade purity of DNA should be sufficient (i.e., unusual purification steps are not required). Digest the DNA in a volume of 100-200ml. DO NOT purify the transgenic fragment – the core facility will perform this purification. Provide the facility with the digested DNA along with an image of an Ethidium bromide-stained agarose gel showing which bands should be purified for microinjection.  BAC DNA should be provided to the core facility in supercoiled or linearized form at high concentration.  Core personnel will dilute the BAC DNA with an appropriate buffer before microinjection.

Note carefully: By far the most robust way to screen initially for transgenic founders is to perform a Southern blot using DNA prepared from tail biopsies of mice. Southern blots are preferable to PCR-based assays at this stage because they unequivocally identify founders and are much less prone to false-negative or false-positive results than other assays. When designed appropriately, the Southern blot can readily provide information about transgene copy number. It can also help to make clear when a founder is a mosaic (with only some of its cells carrying the transgene) or when a founder carries independent chromosomal integrations of the transgene (in such instances, more than one transgenic line can be produced by mating the founder and these lines may differ in expression characteristics). Screens for founders based on transgene expression can accompany Southern blot assays, but ideally should not replace them. Accurate information concerning the success of a microinjection experiment can best be obtained from a Southern blot.

Following the identification of founders (and ideally also their F1 progeny) PCR assays can be established for routine screening for transgenic mice. Ideally, such assays should include internal control reactions. Quantitative real-time PCR assays can also be developed to distinguish heterozygous from homozygous transgenic mice.

The core facility will take every opportunity to keep investigators informed concerning progress on their microinjection experiments. In turn, the facility depends heavily on the investigators for accurate feedback concerning transgenic founder frequencies and germline transmission from ES cell-derived chimeric mice.

Preparation of DNA for Gene Targeting

Linearize 100-200 micrograms of the targeting vector using whatever enzymes are required for the targeting strategy. Precipitate the DNA and wash the pellet with 70% ethanol. Work under sterile conditions from the 70% ethanol wash stage onwards. Air-dry the pellet and resuspend it in sterile TE or sterile PBS at approximately 1 microgram per microliter. Run 0.2 micrograms of the DNA on an analytical agarose gel with appropriate standards to confirm that it has the required form and is at the right concentration. Provide this DNA, a picture of the gel and ideally a rudimentary map of the construct noting drug resistance elements, to the core facility. Ensure that you have made clear the nature of the drug selection strategy and the type of embryonic stem cells to be used.

Note that many gene targeting experiments stall at the screening stage because insufficient prior effort has been invested in ensuring that the screening strategy is robust and effective. If using a southern blot strategy, it is essential to test the probes being used in advance to make certain that they hybridize to bands of the expected size in the absence of excessive background hybridization. A Southern blot strategy usually requires that the probe and the targeting vector should not share sequence in common. The wild-type band detected by the probe should be clearly separated from the mutant band, and the choice of enzyme used for digestion of the genomic DNA should be based on cost and the efficiency of cutting DNA that is not highly purified. Differences of 10 vs. 11Kb or 20 vs. 18Kb may be difficult to detect when working with small amounts of DNA from hundreds of clones. A PCR strategy should be tested using appropriate control plasmids and ideally should involve small amplified regions and highly optimized reactions.

Preparation of ES Cells for Microinjection

Provide the core with a vial of frozen ES cells and any special instructions on how to expand them.  If the core facility has generated the cells in a transfection experiment it performed then it will expand and inject them according to standard procedures.  Investigators may also expand their own cells and provide a healthy single-cell suspension of them to the core facility on the day of injection. The cells should be plated the day before microinjection using healthy confluent cultures as the source (e.g., thaw the vial of cells several days in advance, trypsinize and replate them at least once but, of course, try to avoid extended culture). On the day prior to injection, you should plate several dilutions (e.g., 1:2, 1:3, 1:4) and you may also want to plate the cells with and without feeder cells (using gelatin-coated plastic for the latter). A convenient option is to use a 6-well plate with different plating dilutions and conditions in each well.

On the day of injection, change the medium 2 hours before trypsinization. Choose the well that has the nicest looking culture and trypsinize these as follows:

·          Wash the well with PBS;

·          Add tryspin for 5-7 minutes;

·          Add medium THEN pipet (using a short-form pasteur pipet or a 5ml pipet) to get single cells (but avoid pipeting so vigorously that you start killing lots of cells);

·          Add more medium and pellet the cells by centrifugation;

·          Remove the supernatant fluid and add a small amount (e.g., 0.5-1ml) of fresh medium (avoid old alkaline medium);

·          Pipet briefly but effectively to resuspend the cells, and place the suspension in a freezing vial or an eppendorf microcentrifuge tube on ice (keeping the cells on ice helps to prevent clumping).

It would be wise to double-check with the core facility to confirm details about preparing the cells and when they should be trypsinized on the day of injection.  Try to avoid letting the cells sit on ice for any longer than necessary (ideally, they should be brought to the core facility just before they are ready to be microinjected). Avoid anything that might reduce the viability of the cells – e.g., pipetting too much, or having the cells in very pink medium, or failing to feed the cultures 2 hours before trypsinization.  Avoid an overabundance of feeder cells in the cell preparation – i.e., have the cultures at a good healthy density.  If possible, it makes sense to have two or three ES cell lines ready to go on the day of injection – that way if one of the cultures does not look good, you will have a fall-back.  Also, there are times when we recover larger numbers of embryos than normal – if this happens, and if the injections are going well, you may be able to get two or more clones injected on the same day.

Note: it is the responsibility of the investigator to ensure that the cell suspension will be ready on time. The success of the microinjection experiment depends critically on the health of the ES cell suspension (assuming that the ES cells are otherwise proficient at colonizing the germline).

Common Resource Development Program

The Common Resource Development Program is intended to facilitate the development of mouse strains that are likely to be of general benefit to the diabetes research community.  Examples of such strains would include those that express reporters, recombinases or inducible regulators of transcription in cell types that are of primary significance for diabetes research.  The UCSF Diabetes Center and the DERC will provide financial support for the development and the initial propagation of such strains by investigators who are prepared to characterize them prior to their release to the general scientific community.  Mouse strains covered by this program will benefit from the full assistance of the DERC Mouse Genetics Core Facility. 

Investigators interested in making use of the Common Resource Development Program should send a concise one-page proposal to the Core Director.  At present, there is no deadline for this program.  Proposals will be reviewed by an ad-hoc committee and will be scored on the basis of relevance to diabetes, feasibility, value to the community, and adequacy of proposed plans for analysis, validation and release to the community.

BAC Mutagenesis by Homologous Recombination

Bacterial strains and reagents for manipulating bacterial artificial chromosomes can be obtained from the DERC mouse genetics core facility.  The core facility currently uses the ‘recombineering’ system developed in the laboratories of Drs. Donald Court, Neal Copeland, and Nancy Jenkins at the NCI in Frederick.  This system permits targeted mutagenesis of BACs (and other episomes) by homologous recombination.  Mutated BACs can be used as transgenes or as targeting vectors.  Check here for more detailed information about recombineering and to learn more about how to get permission to obtain the required bacterial strains.  After completing the simplified MTA process described on the website, and receiving permission from the NCI, you may acquire the bacterial strains from the DERC mouse genetics core facility.  Details of additional recombineering plasmids developed at UCSF will be posted here soon.