PREPARATION OF DNA FRAGMENTS FOR MICROINJECTION INTO PRONUCLEI OF MOUSE EMBRYOS

TRANSGENIC FACILITY

Samuel Lunenfeld Research Institute, Mount Sinai Hospital

600 University Ave., Toronto, Ontario, M5G 1X5

416-586-8382

General Considerations

    DNA samples for microinjection should be free of contaminants that might harm the 1-cell stage embryos, such as traces of phenol, ethanol, or enzymes. It is also essential to get rid of any particles that could clog the injection needles. The sample of DNA that are not purified properly will make the injections difficult and/or reduce the survival of zygotes.

    For the above reasons, it is advisable that all solutions added to the DNA sample should be filtered through 0.2 micron filter, and all tubes and pipettes should be rinsed with filtered water prior to use. Glassware with possible detergent residue should be avoided. It is best to use disposable plasticware. Only tissue culture grade distilled water should be used.

    All vector sequences should be removed from a cloned gene before introducing it into the mouse germ line, if optimal expression of the gene is desired.

    Start with Cesium Chloride or Qiagen purified plasmid DNA.

Two alternative methods of purification of the fragment are provided below.

I. Purification of DNA fragment using electroelution followed by NACS* or Elutip** minicolumns procedure (Dawn Bryce, Dong-Li Song, 1994)

To elute the fragment, prepare dialysis membrane by boiling appropriately sized strip in 95 ml H2O + 5 ml 0.5 M EDTA.
Digest 20 - 30 ug of plasmid to separate fragment from the vector backbone.
Use 0.7% agarose gel made in 1X TAE.
Excise band from the gel, insert gel fragment into dialysis bag.
Run gel at 100 V, 10 min or as long as necessary to cause DNA to exit gel slice and line up on dialysis membrane.
Pipette up DNA and transfer to eppendorf tube.
Chill DNA for 5 min on ice, spin 5 min in microcentrifuge to remove any agarose.
Transfer electroelution solution to a new tube.
Use Elutip or NACS mini-columns for final clean-up

NACS Prepac mini-column*

Adjust NaCl concentration to 0.5 M with 5 M NaCl
Purify the fragment using 2 columns for one DNA prep
Hydrate the column with buffer D: 5 ml x 3
Equilibrate the column with buffer C: 5 ml x 3
Keep the column in buffer C
Load the DNA sample to the column and let the solution pass through the column by gravity (slow flow rate is important to maximize binding)
Wash the column with buffer C: 5 ml x 3
Elute the column with buffer D: 100 ul x 4 (let the buffer pass through by gravity and push at the end)
Collect the elute in a 1.5 ml eppendorf tube

The column should never be dried during the above
procedures - it is critical for high recovery

Buffer C: 0.5 M NaCl in TE
(10mM Tris-HCl, pH 7.3, 1 mM EDTA)
Buffer D: 2.5 M NaCl in TE

Spin 5 min in a microcentrifuge
Transfer the supernatant to a new tube
Add 2.5 volumes of 100% ethanol to precipitate
Wash with 70% ethanol x 3, dry pellet briefly
Resuspend in TE to a final concentration

*Elutip** - D mini-column*

(Schleicher and Schuell cat # 27370 available from Xymotech)

Adjust NaCl concentration to 0.2 M with 5 M NaCl
Prepare the column by running through 3 ml of High salt buffer, then 3 ml of Low salt buffer, by attaching the column to 3 ml syringes (follow the instructions)
Run DNA sample through the column at about 1 drip per second using 3 ml syringe - the DNA binds to the column
Wash the bound DNA using 3 ml of Low salt buffer (1 drip per second)
Elute the DNA into a microfugetube using 400 ul of High salt buffer in a 1 ml syringe (1 drip per second)
Add 1 ml 100% ethanol, mix and precipitate overnight at -20 C
Spin down DNA 15 min and resuspend to final concentration (e.g. in 25 ul )

II Purification of the DNA fragment using Geneclean II (Dr. Satoshi Tanaka , 1997)

1. Digest plasmid DNA (20 ug of fragment, in 500 ul reaction mix) x 2 tubes = 40 ug of fragment
2. Precipitate DNA with IPA. Resolve in 50 ul TE each
3. Run on gel (~1.5 x 45 mm well)
4. Cut out gel piece, then put into 14 ml polystyrene tube and weigh (1g of gel ~ 1ml)
5. Add 3 volumes of NaI solution
6. Incubate in 55 C water bath until gel pieces melt (usually ~10 min)
7. Aliquot into 4 eppendorf (1.5 ml) tubes
8. Add 10 ul of glassmilk suspension to each tube
9. Incubate on ice for 15 min. Mix by gentle inversion occasionally
10. Spin for 5 sec at 4 C. Keep on ice. Remove supernatant carefully
11. Rinse with 500 ul of ice-cold NEW Wash buffer (stored at -20 C). Dig pellet with yellow tip to gently resuspend by pipetting up and down. Don't vortex! BE CAREFUL ESPECIALLY WITH THE FRAGMENTS OVER 10 KB
12. Repeat steps 10 and 11 twice more, try to keep samples on ice
13. Add 38-40 ul of pre-warmed (55 C), filtered, 1:10 diluted TE to each of 4 tubes. Incubate for 3 min at 55 C to elute DNA
14. Spin for 1 min , take 35 ul of supernatant, collect to single tube (=140 ul)
15. Spin for 1 min and take the supernatant 3 more times, leaving 10 ul behind each time (eventually, you will have 110 ul)
16. Spin again for 1 min , take upper 60 ul for quantitation and injection, keep lower 50 ul forjust in case.

OPTIONAL (but highly recommended!!): clean up the DNA using Elutip column (see above)

DNA concentration and buffer in which DNA is dissolved

The concentration of the DNA solution that is injected is very important.
At high DNA concentrations (> 10 ug/ml) embryo survival decrease significantly. Injection at low concentrations reduces the frequency of transgenic mice. Therefore, the appropriate quantitation is extremely critical for final result. The sample should be run on a gel with molecular weight markers of known concentration. The best control is to use an injection fragment that has been previously injected with success as THE GOLD STANDARD. Different dilution of the sample DNA and the standard should be made and run on a gel to estimate the DNA concentration of the sample by comparison. The optimal concentration of the sample is > 100 ug/ml.

The buffer used for microinjection contains 5-10 mM Tris (pH 7.4) and
0.1 - 0.25 mM EDTA (pH 5.0) prepared in high quality MilliQ water and filtered.
It is recommended to use this buffer for DNA resuspension especially if the concentration is expected to be lower than 100 ug/ml . However the use of regular TE with all requirements to water and disposable plasticware is also possible.