Services & Costs

For pronuclear injections into the C57BL/6NCr strain, a surcharge of $250 will be charged. We recommend that investigators consider using B6 hybrids such as B6C3 or B6SJL for pronuclear injections as they are more efficient in generating transgenic founders. We mate hybrid females to C57BL/6NCr males, so the founders' background strain is >50% B6 at the outset.

Outside institutions are assessed a surchage to these prices as follows:

• 25% Academic and Non-Profit Institutions
• 50% Commercial Entities

We are currently unable to do cryopreservations for outside institutions.

Pronuclear Microinjection of DNA Constructs
(injection into the pronucleus to create transgenic founder mice):

The number of eggs injected includes ~50 used for an initial test of DNA quality. Re-injection of 100 eggs will be done at no additional charge under certain conditions; a new DNA prep will be required. If embryonic lethality is suspected, the experiment may be terminated before the guaranteed number of pups is generated; half the cost of the injection service will be assessed.

Other strains can be used for microinjection under the following conditions. The investigator will pay for the purchase and housing of 10 stud male and 10 donor female mice. The facility will attempt one day of microinjection at a charge of $750. The injections will continue only if 100 injectable eggs are obtained. Total charges will not exceed the normal charge plus costs associated with purchase and housing of animals.

Injection of Lentiviral Constructs:

3 strains available FVB, C57BL/6NCr (additional charge for pronuclear injection of this strain $250, lentiviral injection no additional charge), B6 hybrid (B6C3 or B6SJL); Other strains available at additional cost.

Blastocyst Microinjection of Embryonic Stem Cell Clones
(generation of knockout or other chimeras):

Re-injection of 30 blasts will be done at no additional charge under certain conditions. Number of clones injected depends on level of service requested. If embryonic lethality is suspected, the experiment may be terminated before the guaranteed number of pups is generated; half the cost of the injection service will be assessed.

Other strains can be used for microinjection under the following conditions. The investigator will pay for the purchase and housing of 10 stud male and 10 donor female mice. The facility will attempt one day of microinjection at a charge of $750. The injections will continue only if 40 injectable blastocysts are obtained. Total charges will not exceed the normal charge plus costs associated with purchase and housing of animals

C57Bl/6NCr strain standard, C57Bl/6 cBrd/cBrd (albino B6) available at additional cost.

Electroporation of Embryonic Stem Cells:

Embryonic stem cells currently must be purchased or obtained in a collaborative agreement by the laboratory. Three commercial vendors are linked off this site under "ES cell vendors." The facility requests as much information as possible on the growing conditions for the cells be provided. The electroporation request form has the details for DNA preparation and amount required. The facility will electroporate the ES cells with the DNA provided; will plate the cells, add selection (positive or positive/negative; we use G418 for neomycin selection, and gancyclovir for negative selection), and pick 150-300 clones. These clones will be replica-plated in 96 well plates; two copies will be frozen, and one will be grown off feeders to provide cells for DNA analysis. Expansion to 24 well plates is provided at an extra charge. The laboratory will identify positive clones, within 1 month. Plates are stored at -80, and delay in identifying clones may result in loss of viability of ES cell clones. The facility will expand up to 5 positive clones; freezing back 3 vials of cells per clone in liquid N2. More clones can be expanded and frozen at an extra charge. The frozen vials will be turned over to the laboratory for storage, unless they are scheduled for microinjection.

Cryopreservation of Mouse Embryos & Sperm
(slow freezing using a ramp freezer or quick freezing by vitrification of 2-cell to morula stage embryos, or sperm, in order to preserve an unused line for future use, or to protect against loss of a line):

The laboratory will provide at least 5 adult male mice, 2-8 months of age, either proven fertile or experienced breeders. The facility will purchase 10-15 young females of the same or a closely related strain, to superovulated and mate with the males. Embryos will be harvested and frozen. The optimal freezing method, as well as the total number of embryos frozen, will be determined empirically by doing an initial test freeze, immediately followed by a test thaw. The percentage of embryos that survive thaw, and then go on to implant and develop into live pups will establish the parameters of the cryopreservation project. If using heterozygous males, the laboratory will be responsible for testing the genotype of the pups resulting from the test thaw.

A charge of $300 will be assessed upon initiation of the project. Total costs may vary if the line proves difficult to freeze, if the investigator desires more than the normal number of embryos frozen, or if the background strain chosen is more expensive to purchase or difficult to superovulate.

PCR Analysis/Genotyping:

Tail DNA Extraction and Analysis of Potential Transgenic Pups :

The facility provides tail snips of potential transgenic pups at 7-10 days of age as part of the micro-injection project. A typical injection project would involve analysis of 20-30 pups.

We will, upon request, isolate DNA from those tail snips, or tail snips provided by laboratory personnel, and provide 500 ul of clean DNA suitable for PCR or Southern analysis. Although we do not routinely quantify DNA isolated by our procedures, 2-10 ul has proven suitable for PCR analysis. The PCR analysis done by the facility is performed one time, with the lab providing primer sequences and parameters for analysis previously worked out by the lab.