Protocols

• Transgene Construct Preparation Protocol
• Transgene Design
• ES Cell Preparation for Blastocyst Injections
• Universal Mouse Genotyping

Transgene Construct Preparation Protocol

The construct should be freed from vector sequences with a restriction digest resulting in fragments that are readily separated by agarose gel electrophoresis. Digest enough material to yield 1µg of construct after purification (yields are typically 25 50%). Perform agarose gel electrophoresis in TAE or TBE buffer. Visualize the DNA fragments in the gel after electrophoresis with ethidium bromide and a minimal exposure to long wave UV light. Ensure no other band overlaps with the transgene band, and excise the gel fragment containing the transgene. Recover DNA from the gel slice utilizing the QIAEX II kit from Qiagen, following the kit instructions EXACTLY. For fragments larger than 10Kb, electroelution before QIAEX may improve yield.

Elute the DNA from the QIAEX resin with 10mM Tris, pH=8.0. Repeat the elution and combine the two eluants. Precipitate the DNA with 1/10 volume 3M sodium acetate, pH=5.2, and 2.5 volumes absolute ethanol. Chill overnight at 20° or for 15 minutes in dry ice/ethanol, and pellet the DNA by centrifugation at 12000 x g (full speed in a microfuge) for 15 minutes. Carefully remove the supernatant with a pipet, and add an equal volume of cold 70% ethanol. Vortex, spin the sample again for one minute, and carefully remove the supernatant with a pipet. Now allow the pellet to air dry.

Add 20 µL of TE or transgene buffer and vortex (transgene buffer is 10mM TRIS, pH=7.4, 0.1mM EDTA). Allow the DNA to solubilize for at least 15 minutes with occasional vortexing. Use 1µL of the construct solution to determine the concentration of the DNA with a fluorometer or low-volume spectrophotometer. Run 200ng of the construct on an agarose gel with appropriate size markers and visualize with ethidium bromide staining. A single, sharp band of the appropriate size should be evident. Attach an original picture of the gel to this report. We will need at least 500ng of DNA for injection, and this should be provided in a tube labeled with the construct name and DNA concentration. The construct name should be short and not contain unusual or Greek characters, subscripts or superscripts. Contact Ted for the electroelution procedure if needed, and a fluorometer is available in his laboratory. A summary of transgene design principles is included for those new to the field.

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ES Cell Preparation for Blastocyst Injections

Help with designing your targeting vector and embryonic stem cell transfection service is offered by the Siteman Cancer Center Stem Cell Core. The MGC will coordinate arrangements with the Stem Cell Core to prepare and transfer your targeted cells on injection days. If you provide your own cells, please use the following protocol to prepare them for injection:

Thaw cells from appropriate positive clone or clones onto a 6cm dish plated with a MEF feeder layer at least 3-5 days prior to injection dates depending on growth speed of your cells. Split cells just before they become confluent, usually 2-3 days before injections, onto three 6cm plates – one for each day of injections as follows: 50% for injection day 1; 30% for day 2; and 20% for day 3.

On the morning of the injections, feed the cells two hours prior to injection time. Just before the scheduled time of transfer, trypsinize the cells for five minutes with pre-warmed trypsin at 37ºC. Place the trypsinized cells in a 15 mL tube, and add an equal volume of ES cell media to inactivate the trypsin. Centrifuge the cells 100-200 x g and remove the supernatant. Resuspend the cells in 1mL of 'injection media' (see below) + 10% FBS. Prepare a second tube containing 10mL injection media + 10% FBS for use on our injection slides. Complete separation of ES cells is essential for blastocyst injections - please GENTLY pipette cells 30-40 times to ensure cell separation.

Bring the cell suspension and extra injection media to us on wet ice.

Injection Media: (available through the tissue culture center on campus)

DME
25mM HEPES without NaHCO3
Osmolarity = 290
PH = 7.4

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Universal Mouse Genotyping

PCR primers are required for all new transgenic constructs or requests for genotyping services. These primers should be 30 nucleotides in length and produce an amplimer of between 100 and 500 nucleotides. Please see our list of primer pairs that work well for common sequences.

This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon, Transgenic Research 12, 521-522 (2003). Materials and procedures are described in complete detail.

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