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Transgenic Resources Program

Resource Services and Contacts | Staff | TRP Forms

EMBRYONIC STEM CELL TRANSFECTION

Construct Preparation
Prices and Guarantees

Embryonic stem (ES) cells are grown on primary mouse embryo fibroblast feeders (PMEF) in the presence of leukemia inhibitory factor (LIF). Approximately 2 x 107 ES cells are mixed with 30-50 µg of linearized construct prior to electroporation. Twenty-four hours after electroporation, ES cells are exposed to antibiotic selection, usually 180 µg of G418. Resistant colonies appear on the plates 10-12 days after transfection. Generally, up to 96 clones are picked and transferred to individual wells of 24-well tissue culture plates and grown up for DNA analysis. Duplicate 24-well plates are made from the same set of resistant clones and frozen. When clones carrying a homologous targeting event have been identified from the first plate, those clones on the stored plate will be thawed and expanded for a bulk freeze and to provide 1,10cm plate per clone for Southern analysis. After the targeting events are confirmed by Southern analysis, these cells will be thawed and used for microinjection.

Note: A common mistake made by first-time investigators is to begin transfection before choosing a proper protocol for determining targeted events. Once the 24-well plates are frozen, thawing and expanding these cells to generate more material for analysis is unwise and generally unsuccessful. The process would involve passaging an unwieldy number of clones, and freezing another set of 24-well plates. Therefore, it is highly recommended that a positive control vector be made if analysis is by PCR. The positive control is composed of the targeting construct extended to include the fragment encompassing the intended primer sequence outside the area of homology.  Thus, the primer set would reveal random integration events of the positive control.  Positive controls will be electroporated at no charge to test the analysis scheme on ten resulting G418 resistant clones.  If a positive control is not available and no correctly targeted clones are identified following the initial electroporation, a fee will be incurred for the resulting excess tech time and reagents needed to help troubleshoot the analysis.

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CONSTRUCT PREPARATION

Each electroporation event uses 30-50 µg of linearized construct. The more DNA available for the electroporation mixture, the more likely it will integrate within the DNA in the host cell. The construct should be at a concentration of 1 µg/ul and free of contamination from ethidium bromide, ethanol, phenol, chloroform, and high salt. Constructs may be delivered to the Health Sciences Building, room T-140 during normal working hours. Ask for Sylvia Roh (slroh@u.washington.edu) or Bob Hunter.

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ES CELL TRANSFECTION PRICES AND GUARANTEES

The charge includes ES cell transfection (20 million cells), selection, clonalisolation, plates for storage at  -80° C, duplicate plates for analysis, thaw and expansion of targeted clones for freeze at -196° C and one 10cm plate per targeted clone for Southern Analysis.  Because different selection vectors included within the targeting construct result in widely variable enrichment for targeted events, no guarantees as to the number of clones arising from electroporation can be made.  However, if 50% or more of the successfully targeted clones are lost through the freezing process the transfection process will be repeated at no additional cost. 

Click here for rates.

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Department of Comparative Medicine
Magnuson Health Sciences Building
Room T-142, Box # 357190
Seattle, Washington 98195-7190
phone: (206) 543-8047
fax: (206) 685-3006

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