Transgenic Research Center In the Cancer Center

DNA Microinjection

We accept linearized DNA or circular BAC DNA for microinjection of one-cell embryos to produce transgenic mice. We ask that investigators follow the facility's protocols to purify their DNA fragments or BACs and submit a picture of the gel showing the purified DNA.
For protocols, see DNA fragment preparation for microinjection or Purification of BACs for microinjection. The facility can also purify your DNA fragment for you at a fee.
We will provide up to 30 pups per DNA construct for genotyping of potential transgenic founders. For DNA transgene fragment purified in our facility, we guarantee a minimum of 2 transgenic founders. We cannot however guarantee mRNA expression and/or germline transmission of the transgene.

The facility have three strain backgrounds available including FVB, B6D2F1 and C57BL6 to accommodate your research needs. Requirements for other mouse strains can be requested for consideration on a case by case basis.

 

Guidelines for DNA fragment preparation for microinjection

The purification of a DNA fragment for microinjection is extremely important. The following protocol provides a guideline for getting DNA that is (1) free of salts, organic solvents, or traces of agarose (2) in the correct and sterile buffer and (3) not sheared or nicked.

MiTE (microinjection TE buffer):
10 mM Tris-HCl, pH7.4
0.1 mM EDTA
autoclaved and filter (0.2um) sterilized.

A large prep of the plasmid DNA can be made and purified by cesium chloride (CsCl) gradient or a Qiagen column.
The fragment of interest is then isolated from vector sequences by restriction enzyme digestion, removing as much of the vector sequences as possible.
Fractionate the digested DNA in an agarose gel by electrophoresis using 1X TAE buffer containing ethidium bromide (0.5 ug/ml).
View the DNA fragments using a long-wave UV light and carefully excise the fragment of interest using a fresh, sterile scalpel. Dice the agarose block and transfer to one or more 1.5 ml eppendorf tubes depending on the size of the gel slices.
Further clean the DNA fragment by using a Qiagen QiaexII gel extraction kit (Qiagen Cat #20051) or Geneclean method using a Geneclean Kit (Bio 101, Inc. Cat #1001-200). Elute DNA with MiTE (10mM Tris-HCl, 0.1mM EDTA, pH7.4).
Filter the DNA through a small 0.45um Millipore spin filter (Millipore Cat #SLHV004NL). We can also filter the DNA for you for a $25 fee.
Quantitate the DNA concentration by either a fluorometer with calf thymus standards or by comparing an aliquot of the DNA prep with size standards of known concentration on an agarose gel.
Give a total of 1 ug DNA at 50 ng/ul or more in an eppendorf tube to the facility for microinjection. Attach a picture of an agarose gel showing an aliquot of the purified DNA fragment and its concentration.

Preparation of BAC DNA for microinjection

Polyamine microinjection TE buffer (PMiTE) for BAC DNA:

1000x Polyamine Stock

30 mM Spermine  (Sigma, tetrahydrochloride, #S-1141)
70 mM Spermidine (Sigma, trihydrocholoride, #S-2501)

Dissolve the spermine and spermidine together in autoclaved distilled water, filter sterilize (0.2 micron filters), and store at -20 C. Since the polyamines are very hygroscopic, it is suggested that small quantities (1 gram) should be ordered and then all of it should be prepared at once.

Polyamine Microinjection TE Buffer (PMiTE)

For 50 ml:
10 mM Tris-HCl,  pH 7.5  0.5 ml of 1 M Tris-HCl,  pH 7.5 (autoclaved)
0.1 mM EDTA, pH 8.0  10 microliters of 0.5 M EDTA, pH 8.0 (autoclaved)
100 mM NaCl   1 ml of 5 M NaCl (autoclaved)
1x Polyamines 50 microliters 1000x Polyamines mix
Autoclaved H2O up to 50 ml

NOTE: Prepare fresh and discard unused polyamine microinjection TE buffer.

1. Make a large CsCl prep of the BAC DNA.

2. Estimate the DNA concentration by digesting an aliquot of the prep with either SalI or NotI and run the digest on a regular gel. The concentration can be determined by comparing the 7 kb vector fragment with a range of lambda standards.

3. Digest 10 ug of BAC DNA with restriction enzyme (usually either SalI or NotI) and heat inactivate the enzyme at 65 degrees C for 20 minutes.

Skip this step if circular BAC DNA is desired to be injected.

4. Buffer exchange with Centriprep-30

To prepare the DNA for microinjection, multiple steps of buffer exchange are performed with Centriprep-30 concentrator columns (Amicon, Beverly, MA, CAt#4306).

Read and follow the instructions included with centriprep-30.

All steps must be done at 4 degrees C.

(4A). To remove trace glycerol present in the membrane of centriprep-30, rinse the column by adding 10 ml PMiTE to the outside chamber of the column. Spin at 1500g (2800 rpm) in a Beckman JA-10 rotor with inserts for 10 minutes. If use a different rotor, calculate the correct rpm using the centriprep instructions.

Decant both inside and outside chambers.

(4B). Repeat step (4A). The column is now rinsed and ready to use.

(4C). Mix DNA with 15 ml of PMiTE and add it into the outside chamber of the column. Spin at 1500g for 25 minutes. Dispose the inner chamber solution. Repeat spin and disposal of inner chamber solution another two more times.

(4D). Add 10 ml of PMiTE to the outside chamber and spin at 1500g for 20 minutes. Dispose inner chamber solution and spin again for 15 minutes. Dispose inner chamber solution.

(4E). Add 5 ml of PMiTE to the outside chamber. Spin at 1500g for 15 minutes. Dispose inner chamber solution. Repeat spins of 5-10 minutes followed by removal of inner chamber solution until outer chamber solution is less than 0.5 ml.

5. Filter the DNA through a small 0.45um Millipore spin filter (Millipore Cat #SLHV004NL).

6. To determine the integrity of DNA, run approximately 200 to 500 ng of DNA on a pulsed-field gel (estimate a 50% yield after buffer exchange columns).

7. To quantitate the concentration of DNA, digest DNA with either SalI or NotI and run digest on a standard 0.8% agarose gel. The concentration can be determined by comparing the 7 kb vector band with lambda standards.

8. Give a total of 1 ug BAC DNA to the facility. Attach pictures of both pulsed-field gel and regular agarose gel showing the integrity and concentration of the prep.

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