Fees for Service

In addition to the core service of blastocyst injection and transfer, the Center is able to provide assistance in the electroporation of targeting vectors and expansion of targeted ES cells. For these services, contact Robin Nguyen (x56289) or by email at robin.nguyen@utsouthwestern.edu to inquire about availability.

Electroporation and Selection of ES Cells                               $2,000

Investigator’s targeting construct (50µg) will be electroporated by the Transgenic Technology Center (TTC) into 1 x 107 wt-ES cells from the 129/SvEvTac (SM-1 cells) or 129X1/SvJ x 129 S1/Sv - P (R1 cells) or 129 S7/SvEvBrd (AB2.1 cells) strain. To use either R1s or AB2.2s, a Materials Transfer Agreement must be signed. UTSW’s Contracts Management (x84319) has the appropriate forms. After selection, 5 96-well plates of colonies will be picked from the electroporation. Stock plates of these clones will be frozen and the Investigator will be provided with two 96-well replica plates of each stock plate for DNA isolation. Charge includes purchase of ES cells supplied by TTC.

Expansion of Positive ES Cell Clones                                       $1,250

A maximum of 5 positive ES cell clones will be thawed and expanded and, three 35 mm2 dishes will be frozen per clone.

Preparation of ES Cells for Injection                                        $500

TTC cultures Investigator’s targeted ES cells and prepares them daily for blastocyst injections during the scheduled injection week.

Anticipated Services

It is anticipated that during the next 6 months, the Center will expand its capabilities and services offered to generate gene knock-outs. These services will include:

1. Construction of targeting vectors.
   
   
2. Screening of ES cell clones for recombination – Southern Blotting.
   
   
3. Screening for mycoplasma contamination.
   
   
4. Karyotyping of ES cell clones.
   
   
5. Provide select targeting vectors.

Investigator Requirements

ES Cell Electroporation

1. IDR for $2,000 payable to the Transgenic Technology Center, MC 8816.
   
   
2. Linearized isogenic targeting vector in sterile water at a concentration of 1 µg/µl. For constructs £17 kb, 50 µg is needed; for constructs >17 kb, 100 µg is needed.
   
   
3. Gel picture of linearized targeting vector run alongside uncut targeting plasmid to demonstrate complete digestion.
   
   
4. Map of targeting vector indicating arms of homology (size and cloning site), site of linearization, and selection cassette(s).
   
   
5. Completed Gene Targeting Request Form.

Timeline for Gene Targeting

Day 1                     Thaw feeders

Day 3, AM               Split/Irradiate feeders

Day 3, PM               Thaw wt-ES cells

Day 6                      Electroporation

Day 7-18                 Selection with G418 and Gancyclovir

Day 19-20               Pick colonies (5 x 96-well plates), each plate divided into 3 plates,

                               1 Master plate and 2 replica plates

Day 23-24               Freeze stock plates

Day 33                    Investigator receives 10 replica plates for DNA isolation
                               (2 plates/96-well plate picked)

Times are estimates only and may be shorter or longer depending on individual circumstances.