|
Transgenic Resources Program
Resource Services and Contacts | Staff | TRP
Forms
PRONUCLEAR MICROINJECTION
Pronuclear microinjection involves injecting a small quantity
of DNA into the pronuclei of recently fertilized one-cell
mouse embryos. The standard background strain used for
the embryos is C57BL/6 x C3H, abbreviated B6C3. Embryos
are produced by mating B6C3 females to C57BL/6 males,
generating potential founder animals which are 75% C57BL/6
and 25% C3H. After injecting the embryos with DNA, the
surviving embryos are transferred surgically to the oviducts
of pseudopregnant females. Embryo transfer may occur on
the same day of injection, or following in vitro culture
to 2-cell overnight.
The service includes: the purchase and housing of the animals
used to produce embryos, the cost of hormones to stimulate
ovulation in donor females, the cost of providing pseudopregnant
recipients for embryo transfer, and technical expertise.
The investigator begins paying per diem cage charges
on the day of embryo transfer. The total number of cages
generated from one injection day can range between 1
and 4, with recipients typically housed in pairs. Gestation
length is approximately 20 days. Tail (or ear) biopsies
for genotyping purposes are taken from the pups between
3 and 4 weeks of age, and the pups are weaned between
4 and 5 weeks of age. Therefore, the total length of
time from embryo injection to tissue biopsy is approximately
7 weeks. The investigator will be notified when the biopsies
are available to be picked up from the small freezer
located in HSB, room T-140. Please take only the samples
labeled with the investigator's name and sign off on
the sheet posted on the freezer for this purpose.
Top
CONSTRUCT PREPARATION
Expect higher integration
rates if the construct is linearized. Removal of the plasmid
backbone is thought to be beneficial. The construct must be physiologically
benign. It should be free of phenol, chloroform,
ethidium bromide, high salt and ethanol. If the
final purification steps are followed with this
in mind, almost any purification method will
be successful as long as some sacrifice is made
for quality over quantity. The
concentration should be at least 50 ng/µl. The higher the concentration
the better the results. The concentration is then measured using
a fluorimeter. The construct is diluted to a final injection
concentration of 2 ng/µl. Please deliver constructs to
T-140, Health Sciences Building. Ask for Bob Hunter or Sylvia Roh.
Prior to pronuclear injection, the diluted construct is spun
through an ultrapure 0.45µm filter. This technique removes
any residual aggregates that might clog the needle during
injection.
Top
PRONUCLEAR MICROINJECTION
PRICES AND GUARANTEES
-
Per
injection day 70 or more viable, injected
embryos will be transferred to the pseudopregnant recipients. If
fewer then 70 are transferred, the construct will be injected again
at no additional charge.
-
In the event that an alternate
host embryo strain is required, the additional
costs incurred from purchasing and housing a special
strain of stud males, will be charged to the
investigator. The
investigator will also pay the difference in
purchase price between the cost of B6C3 females
typically used, and the cost of the embryo host
strain selected. No guarantee
will be made as to the number of injected embryos
transferred, due to strain variability in embryo
production.
- C57BL/6 embryos
are
available at no additional cost per injection
day, but without guarantee. This
strain is less efficient. Thus three injection days
are
typically required
to achieve the same results as one day of B6C3
injection.
- Cancellation
of injection within two weeks of the scheduled
date will result in a $100 fee to help cover the cost
of the embryo
donor mice purchased
for the injection.
Rates |
