About the Core
The Transgenic Core generates transgenic and gene knockout mouse models relevant to obesity, diabetes and nutrition research. The Core also provides consultation on all aspects of making transgenic and gene knockout mice.
Generation of Transgenic Mice.
DNA transgenic constructs, supplied by the investigator, are microinjected into fertilized one cell embryos. Tails from potentially transgenic founders are then supplied to the investigator for genetic analysis. Identified founders are then provided to the investigator.
FVB/N Mice: $2,000*.
C57BL/6 Mice: $3,200*
Repeat injection of new DNA prep:
* cost does not include per diems, shipping, etc.
Specifics / Guarantees with regards to Generation of Transgenic Mice:
FVB/N OR C57BL/6 TRANSGENICS:
Service: Pronuclear stage zygotes are injected with the DNA construct. The DNA is diluted until we have a ratio of approximately 70% 2-cells (normal development) and 30% 1-cells (delayed development) after injection and over-night culture. Historically, this ratio gives us reasonable in vivo survival (30%) and consistently gives us founders. Once we have achieved that 70/30 ratio, we will inject enough embryos to generate 40- 50 offspring. Assuming an average 10% incorporation rate, this usually gives 4-5 founders.
Guarantee: 4 founders* *See below.
*REPEAT FVB/N OR C57BL/6 DNA INJECTIONS WITH NEW PREP
Service: After we have generated 40-50 offspring from injections which resulted in the 70/30 ratio, if there are no transgenics we will ask for a new prep and charge the new prep fee. Note: If we only inject the injection buffer, virtually 100% of the embryos will be 2-cells after overnight culture. So if we achieve the 70/30 ratio and do not get transgenics, we assume that some contaminant in the DNA prep is delaying the embryos. That is why we request that the DNA prep be as pure as possible.
Guarantee: 4 founders
Gene Targeting – Generation of Chimeric Mice from Targeted ES Cells.
Targeted ES cells are injected into 3 day old embryos (blastocysts). Highly chimeric mice are then provided to the investigator.
Cost: Injection of 2 ES cell clones: $650*.
* cost does not include per diems, shipping, etc.
Specifics / Guarantees with regards to Generation of Chimeric Mice from ES Cells:
Service: 20-40 Blastocysts are injected with 1-2 ES clones provided by the investigator and transferred into 2-4 recipients. We set up enough mice to produce 40 blastocysts. About 5% of the time we do not get 40.
Guarantee: If we have less than 20 blastocysts we will still inject but will not charge. If the mice fail to maintain a pregnancy or the number of births is very low (1 or 2 pups/litter) we will not charge. Under normal circumstances, the success of the ES cell injections is dependent on the quality of the ES cells. Since the ES cells are provided by the investigators we cannot guarantee high percentage chimeras, or even chimeras. So if we get litters which survive but no chimeras we will still charge.
Cryopreservation of Mutant Mice.
Fertilized once cell embryos are cryopreserved (contact Transgenic Core for details).
Cost: Embryo freezing: $3.50 per embryo (it is recommended that investigators freeze a least 100 embyros)
Service: Freezing a number of embryos determined by the investigator and storing those embryos in liquid nitrogen. The charge is per embryo and we recommend freezing at least 100. Investigator maintains the colony and provides us with mouse oviducts after superovulation and mating. We provide training to cut out the oviducts of the mice, hormones to superovulate, and medium to put the embryos into. We take the oviducts, extract the embryos and freeze.
Guarantee: We only charge for embryos that we freeze. Often, the first time the males are mated, they do not fertilize. Also, with inbred transgenic strains the reproductive efficiency may be low and we are prepared to do several reps, if necessary in order to generate the desired number of embryos. If the investigator contracts for 100, we usually try to collect more than 100 and perform an embryo transfer to determine freezing success, leaving at least 100 banked down.
Generating Mice from Frozen Embryos.
Service 1: Embryos (frozen) from outside sources (after clearance from the Animal Research Facility) will be thawed and transferred into pseudopregnant recipients.
Guarantee: We will only charge the full price if live offspring result. Since the embryos are from an outside source we cannot guarantee that they were viable when we received them. Historically, the quality and viability of frozen embryos from outside sources has varied greatly. If frozen embryos are not viable after thaw and we were going to use extra recipients from our DNA or ES cell projects we will not charge. If the frozen embryos are not viable and we have set up extra mice for recipients, we will charge the cost of the mice. If the embryos appear to be viable (thaw correctly) and do not survive, we will try again if we originally received more than one vial or straw of embryos for no extra charge. If we achieve the same result we will charge the cost of the recipients, if applicable.
Service 2: Embryos previously frozen within our lab will be thawed and transferred into pseudopregnant recipients.
Guarantee: Live young (at least one breeding pair) or no charge.
In Vitro Fertilization
Service: B6D2F1 hybrid eggs will be inseminated with frozen or fresh sperm (pending import approval by the Animal Research Facility). If fresh sperm is used, we will train investigators to cut out the tail of the epididymus. The resulting embryos will be transferred into pseudopregnant recipients.
Guarantee: We will only charge the full price if live offspring result. Since the sperm is from an outside source we cannot guarantee that it will be viable or motile when thawed or extracted from the epididymus. If fertilization rates are low or non existent we will only charge for the cost of the mice used to generate oocytes. If the sperm appears to be viable and capable of forward progressive motility and we do not get embryos or there is no resulting offspring, we will try again if there is additional sperm and will only charge for the cost of the females used if on a second try there is no live young.
Consultation on all aspects of transgenic and gene knockout work is provided.