Transgenics & Knockouts Transgenics

All work done by the microinjection lab requires an IACUC code (Institutional Animal Care and Use Committee). For Instructions

Once you have received an IACUC code, a request must be made in Internal Services >Transgenic and Embryo Manipulation Lab. Fill in all fields. *Item select # 1. Production of Transgenic Mouse (DNA Microinjection)

Instructions for Preparation of DNA for Microinjection

Note: DNA for pronuclear microinjection must be prepared according to the instructions below. DNA of poor quality or low concentration will not be injected.

General Considerations:

The quality of the DNA preparation used in microinjection directly affects the efficiency of DNA integration. Each of the following parameters will affect the efficiency of DNA integration:

1. The injected fragment must be free of flanking plasmid sequences (which may be toxic). Therefore, the construct should be designed so that the insert for microinjection can be excised without vector sequences.
2. The DNA must be intact, without accompanying degradation to prevent integration of partial constructs.
3. The DNA must be free of contaminants such as phenol, ethanol, enzymes, etc., which may harm the embryo.
4. Solutions used in DNA purification and dilution buffers should utilize fresh, high quality sterile reagents, and Water for Embryo Transfer (Sigma W1503) must be used.
5. It is essential that the DNA solutions do not contain any particulate material that may clog the microinjection needles.
6. DNA concentration must be measured exactly in order that the final concentration for injection (1 - 3 ng/ul) can be accurately determined. The final dilution of DNA in injection buffer will be done out by personnel of the transgenic facility.
7. 50 ul purified DNA at a high concentration (50-100 ug/ml) should be delivered to the transgenic lab ten days before the assigned microinjection session to check for DNA purity.
8. It is highly recommended that you prove that your construct is expressed in vitro by an appropriate cell line. If this has not been shown for your construct, state it in your application.

Preparation of Plasmid DNA:

In order to ensure optimal efficiency, the following procedures must be followed in preparing DNA for microinjection. DNA that has been prepared in a way different from the above conditions will not be accepted for microinjection by the transgenic unit.

1. Isolate plasmid DNA by any conventional technique.
2. Digest ~50 ug of plasmid with appropriate restriction enzyme, removing as much vector sequences as possible from the insert as they could inhibit the expression of transgene and may be toxic to the zygotes. Run a minigel to check the digest.
3. Separate the insert from the vector on 0.8% agarose gel run in TAE. Cut out the band of interest using a clean razor blade and transfer the gel slice into a DNase-free Eppendorf tube, minimizing DNA exposure to UV light.
4. Purify the insert by one of the following methods:

• QIAquick PCR purification kit (Qiagen 28106)
• Electroelution, ethanol precipitation, ion exchange column (e.g. Elutip-D mini-column Schleicher and Schuell 462615 or Xymotech 27370)
• GENECLEAN kit (Q.BIOgene 1001-200)
• NucleoSpinTM Extract Kit (Clontech K3051-1)
• QIAEX II gel extraction kit (Qiagen 20051)

It is strongly recommended to use powder-free gloves when handling DNA preparation to avoid potential clogging of injection needles.

1. Precipitate the DNA in ethanol. Dissolve it in a small volume of embryo-tested water (Sigma W1503, available from Chemical Stores), which has been filtered through a 20 um filter (DO NOT use filters which have been sterilized with ethylene oxide such has Schleicher and Schuell. Recommended filters are Whatmann 0.2um available from stores #010005432).
2. Centrifuge 15 min in an Eppendorf centrifuge to remove particulate material. Measure OD from this supernatant. Store DNA at concentrations >200 ug/ml at –20°C.
3. Run a minigel with molecular weight markers of known concentration, such as DNA mass ladder, and compare staining intensities. Check the purity of the DNA, make sure that it is intact, of right size and there is no smear of sheared DNA.
4. Submit at least 50 ul of the prepared DNA at a concentration of 50 - 100 ug/ ml, together with the picture of the gel, to the TG Unit ten days before the assigned microinjection session, to be stored at 4°C before use. Label the tube with the construct name, concentration, investigator and date. The DNA will be checked for purity. DNA containing particulate material will not be injected.

DNA that has been prepared in a way different from the above conditions will not be accepted for microinjection by the transgenic unit.

Preparation of BAC DNA:

Prepare BAC DNA using the NucleoBond ® BAC 100 purification kit. The NucleoBond ® BAC 100 purification kit (MACHEREY-NAGEL Germany) is available from Biolabs, Jerusalem. One prep should yield 40 -50 ug DNA.

Be very gentle through the whole procedure. Do not do any vigorous mixing or shaking. For BAC DNA, use only cut (wide bore) tips.

From experience we recommend the following adaptations to the protocol provided with the kit:
Step 4: Use option 1, and do not spin before filtering. Use water, not buffer N2 for wetting the filter.
Step 8: Precipitation in a swinging rotor is better. If your rotor cannot reach 15,000g, spin at lower velocity for an hour.
Step 10: Dissolve in 150 ul at 37°C for 1 hour. Collect the DNA in solution to a fresh tube. Add a further 100 ul to the original tube and incubate overnight at 37°C.

BAC Preparation for Microinjection:

BAC DNA must be linearized and checked for integrity before microinjection.

Linearize with a rare cutting enzyme whose sequence is present only once in the BAC DNA, usually PI-SceI. Following linearization dialyze against BAC injection buffer. Place injection buffer (available from the microinjection lab) in a petri dish and load the BAC DNA onto the centre of a 0.025 um VSWP membrane (Millipore #VSWP02500, available from the microinjection lab) for 3 hours.

Run ~ 0.5 ug using Pulse Field Gel Electrophoresis.

The microinjection lab requires 500 ul DNA at a concentration of 3ng/ul.