|Lentiviral RNAi Protocols - Transgenics|
|Transgenic Animals via Lentiviral Injection||
Perivitelline Injection Protocol
Purpose: To generate transgenic mice.
Engineered lentiviral particles are microinjected directly into the perivitelline space of mouse embryos 0.5 days after fertilization.a,b The viral particles are comprised of a self-inactivating viral vector containing a gene of interest and a promoter, as well as a marker gene in some cases. The particles also contain reverse transcriptase to catalyze the incorporation of the vector sequence into the genome and the viral particle itself has a glycoprotein coat that mediates its adherence to the embryo. In some percentage of cases, the viral vector incorporates into the genome of the one-celled embryo, carrying the gene of interest with it. Once integrated, the viral sequences cannot be replicated, due to a deletion in the requisite sequence. Embryos are incubated at 37°C overnight. Two-cell embryos are implanted into the oviduct of pseudopregnant female mice the following day. Pups resulting from this procedure are genotyped to test for the presence of the transgene.
1) Three days prior to lenti-viral injections, administer 0.1 ml PMS
by intraperitoneal injection to female donor mice (C57BL/6 or FVB).
1) The afternoon prior to injection day, set up two 60 mm culture dish
(Corning catalogue #25382-381) with four 50 ?l microdrops of KSOM (Cell
and Molecular Technology, catalogue #MR-101-D) covered with oil, and one
35 mm dishes (Corning catalogue # 25382-348) with three 50 ?l microdrops
covered with oil. Label each dish with your name and the date, then place
into the CO2 incubator. The media is incubated overnight to allow the
temperature, and more importantly, the pH to equilibrate.
1) On the morning of injection, sacrifice female donors in groups of
five or less, using cervical dislocation.
1) Viral aliquots provided by the investigator are kept at –80
C. Take one aliquot of 5-10 ?l and thaw within a 50 ml conical tube filled
with ice. Keep the conical tube in an ice bucket. Any direct contact with
the virus should be done under the hood while double gloved.
Setting up the microscope:
Purpose: To transfer mouse 2-8 cell embryos into 0.5 day pseudopregnant recipient females for any of several procedures; embryo transfer rederivation, subsequent to in vitro fertilization, and following pronuclear injections and cryopreservation recovery.
1) Pipette three microdrops (~50 ?l) of FHM or M2 media on the bottom of a 30 mm petri dish.
2) Cover the media with embryo-tested mineral oil and leave at room temperature.
3) After weighing a recipient female, inject the calculated dose of Avertin intraperitoneally.
4) The caudal dorsal area of the anesthetized female is shaved, then scrubbed using povidone iodine solution or scrub alternately with 70% alcohol on cotton tipped swabs (repeat two times).
5) Place the prepped pseudopregnant female under the dissecting microscope, ventral side down.
6) Surgical instruments should be either autoclaved or bead-sterilized. Using a small scissors and watchmaker forceps, make a 5 mm skin incision. Separate the skin from the body wall by blunt dissection using scissors tips. Make an incision through the body wall avoiding nerves and large blood vessels. Manipulate the incision until the white fat pad surrounding the ovaries is visible. Grasp the fat pad with a dull forceps and pull it through the incision.
7) Position the ovary for easy access to the oviduct.
8) Pipette enough embryos for one recipient into a microdrop of FHM.
9) Load a transfer pipette (the tip diameter should be only slightly larger than the embryos) with oil from transfer dish by using mouth suction. Oil level should be close to the beginning of the widest part of the pipette. Next, draw a 2-5 mm air bubble into the pipette, then media with embryos. Carefully press pipette barrel into the clay on the dissecting microscope until ready to transfer the embryos.
10) Gently tear open the bursa surrounding the oviduct with two pair of forceps allowing access to the infundibulum, which is the opening to the oviduct. In case of bleeding, use a sterile cotton swab to gently blot.
11) Once the infundibulum can be visualized, slide the tip of the loaded pipette in and blow into the pipette until the air bubble is visible within the oviduct.
12) Check the pipette tip underneath the microscope to make sure the embryos have been transferred.
13) Gently replace the ovary/fat pad and uterus into the abdominal cavity.
14) Close the body wall with one or two simple interrupted sutures of 5.0 silk. The skin is apposed with one or two sterile surgical clips.
15) The transfer can be done either uni- or bi-laterally. The pups are
expected on day 20 following surgery.
|This webpage was created by Chris Dillon|