July 27, 1999

DNA Vaccination of Mice

Conversation with Ron Haskell (Davidson Lab 3-5574)

Vectors: from the Krieg lab (U of Iowa). Contact Ryan Kniewell 5-6842.

pMCG16 and pMCG50 derived from pUK21-A2 which was derived from pUC-19. It has a Kanomycin Resistance marker and a CMV promoter. The Wilson lab has primers that are designed to sequence from the ends of the multiple cloning site (MCS) called, pmcgTOP and pmcgBottom. The primers are located in the freezer box labeled Ryan K, General stores.

The sequence file from GenBank for these two vectors contains a Hepatitis B surface antigen in the MCS that was inserted as an EcoRV – PstI fragment. A NotI/BglII double digest will excise a 700 bp band. The accession numbers are:

pMCG16s = AF053408 and pMCG50s = AF53409.

Mouse injections and bleeding schedule:

For each construct order 4-5 Balb C female mice that are no more than 4 weeks old.

- younger mice have better immune responses.

The day the mice arrive, take a retro-orbital bleed (see below) for pre-immune serum, and before the mouse awakes, intradermally inject 50 – 100 micrograms of DNA (resuspended in either TE or PBS) into 6 – 10 locations in the mouse's back. When injecting, look for the skin to bubble and then move to a new location.

At two weeks, take another retro-orbiatl bleed, and administer a 50-100 microgram booster of DNA.

At four weeks, take another bleed and administer another booster.

Test this serum for antibodies against your protein (see below).

Once antibodies are detected, continue to boost every two – four weeks. And bleed the mouse for antibodies as often as three times per week.

Saccrafice the mouse prior to 18 weeks to reduce high background in the later bleeds.

Retro-orbital bleeds:

Prepare a metaphane (inhaled anesthetic) soaked gause plug in a 50 mL Conical Tube (remember to pour un-absorbed metaphane back into bottle).

Insert mouse's head into the metaphane-Tube until the mouse falls asleep.

Using a sterile 6 inch small bore pasteur pipette, insert pipette tip into the eye socket and behide the eye. Suction blood until the tapered end of the pasteur is full (~200 micorliters). Dispense blood into a labelled epindorf tube.

Preparation of Serum:

Typically, a 200 microliter bleed should yield approximately 100 microliters of serum.

Allow the blood to sit for either 2hrs at room temp or overnight at 4 C to clot.

Spin the clotted blood at 5000 x g for five minutes to separate the clot from the serum.

Using a p200 pipettman, carefully aspirate the serum (supernatant) from the clot (pellet).

Alliquot the serum into epindorf tubes as desired and freeze at – 20 C.

Testing serum for antibodies:

You should test the serum for antibodies against your protein using the assay for that you intend to use the antibodies. Western blots, IFA's, IP's, etc.