Fees for Service
The UTSW TTC injects and transfers a minimum of 200 blastocysts per construct to pseudopregnant females. The Center will care for the pregnant females and litters for two weeks after birth. Litters are then transferred to the Investigator for breeding. In the event that no chimeras are produced, the Investigator should meet with Center staff to review protocols and discuss options.
Injection of ES Cells $1,750
Guarantee Policy
In exchange for the fees charged for services, the Center warrants that it will produce at least 3 mid to high percentage chimeric animals for the Investigator whose ES cell preparation fulfill the criteria summarized below.
Investigator Requirements
1. The Investigator prepares targeting vector, electroporates ES cells, and confirms successful targeting of construct. Investigator amplifies ES cell clones with targeted gene knockout. ALL OF THE FOLLOWING ARE REQUIRED TO BE PUT ON THE WAITING LIST:
a) Expanded and frozen ES cell clone(s) (A minimum of 3 expanded clones are
recommended)
b) IDR for the appropriate cost
c) Completed Generation of Chimeric Mice Request Form
2. The Investigator must call the Center (x56287) to reserve a date for blastocyst injection and to discuss details about the preparation of cells for injection.
3. The Investigator must prepare ES cells for injection as follows. A detailed protocol is available from the Center.
a) Passage cells once (preferably two days) before the day of injection.
b) Thaw cells by Thursday of week before scheduled injection date.
c) Feed ES cells 1 hour before trypsinization.
d) Pre-plate ES cells to remove feeders.
e) Resuspend cells in appropriate volume of fresh ES media.
4. The Investigator must deliver ES cells on ice to ND11.223 at Noon on the injection day.
5. The Investigator’s ES cells must be deemed viable as determined by morphological examination by Center personnel in order to be injected. If the cell preparation is of poor quality, the Center staff will meet with the Investigator staff to discuss strategies for correcting the problem. The most common problems with ES cell preparations are:
a) Vigorous mechanical disaggregation leading to a high percentage of lysing cells.
b) Excessive feeder cells in preparation leading to difficult and time consuming
injections.
c) Too short a period of enzymatic disaggregation leading to clumps of ES cells.
6. It is strongly recommended that the Investigator certify ES cell cultures are free of bacterial and mycoplasma contamination.
Time Line for Generation of Chimeric Mice
Day 0 Set up matings of superovulated C57Bl/6 females with stud males.
Day 1 Set up matings of ICR female mice with vasectomized ICR males to
generate pseudopregnant recipients.
Day 4 0800 Harvest day 3.5 blastocysts from uterine horns of donor C57Bl/6 females.
1400 Obtain ES cell preparation from Investigator. Inject 10-12 ES cells into
blastocyst cavity of Bl/6 embryos.
1600 Transfer 10-15 injected blastocysts into uterine lumen of day 2.5
pseudopregnant recipient.
Day 21 Litters born naturally or C-sectioned and fostered.
Day 32-35 Assess coat color of pups to identify chimeric mice. Grade coat color based
on level of ES contribution: 0-100%.
Day 35-42 Transport chimeric mice to the Investigator.
Day 42 Investigator must sex and wean.
Day 56 Breed F0 chimeric male mice with wild-type C57Bl/6 females.
Day 76 Birth of F1 pups.
Day 83 Check coat color of F1 mice for agouti. Screen F1 agouti pups for the
presence of mutation.
Day 97 Wean F1 pups.
Day 110 Breed heterozygous F1 male with F1 females.
Day 137 Birth of F2 pups.
Day 151 Wean F2 pups. Screen for homozygous knockout mice.
Day 165 Breed F2 male with F2 female to generate mutant line.
