Biology Reference
In-Depth Information
1.
Usually, an overnight bacterial culture in 100 mL LB broth with ampicillin yields
enough DNA for microinjection to create transgenic mice. Prepare DNA with
Maxi (Qiagen) following the manufacturer's protocol.
2.
The DNA to be microinjected should be free from the vector sequence. Digest
the plasmid with
EcoRI
and
PstI
. The transgene containing the promoter,
enhancer, and reporter gene are then purified by electrophoresis on an agarose
gel. After ethidium bromide staining, the transgene fragment is eluted from the
gel slice using a Gel Extraction Kit (Qiagen). The DNA fragment should be
resuspended at concentrations of about 5 ng/
µ
L in TE (10 m
M
Tris HCl pH 7.5,
0.25 m
M
EDTA).
3.
Microinject about 5 pL of the purified DNA into the pronuclei of fertilized mouse
ova
(15
,
16)
. Sacrifice the pregnant female mice at the desirable stages of gesta-
tion to obtain embryos. Embryos older than 13.5 dpc, may require the dissection
of tissues to allow full penetration of the fixing and staining solutions.
4.
Place the embryos or tissues into six-well tissue-culture dishes on ice. Wash them
with cold PBS once, and fix with 4 % paraformaldehyde for 20 min on ice with a
gentle rocking motion to increase penetration of the fixative.
5.
Aspirate the solutions, paying attention not to damage the embryos. Wash with
PBS for 10 min on ice while rocking. Wash the samples two more times with PBS.
6.
Add the X-gal staining solution. Put the cover on the dish and seal it with Saran
wrap. Incubate the samples for 1 to 24 h at 30
°
C in the dark. The levels of the
staining depends on the expression level of the
β
-galactosidase gene. (
See
Note 9
.)
7.
After staining with X-gal, wash the samples briefly with PBS. Fix the samples
again with 4% paraformaldehyde to prevent the leakage of X-gal stain.
8.
The X-gal stained tissues can be sectioned for histological analysis. Dehydrate
them with alcohol and embed them in paraffin wax. Counterstaining is useful for
identification of cell types expressing the
β
-galactosidase gene. (
See
Note 10
.)
9.
The presence of the transgene in embryos can be tested with DNA isolated from
placentas
(8)
. PCR amplification of genomic DNA with specific primers can be
used for rapid detection of the transgene
(17)
. Southern blot analysis can be
performed to determined a copy number and integration sites of the transgene.
(
See
Note 11
.)
4. Notes
1.
PBS can be substituted for HBSS.
2.
β
-gal is a convenient reporter gene to identify the specific cells that express
the transgene. However, if the experimental goal is to quantitate small differ-
ences between different constructs, then CAT or luciferase reporter systems
are required.
3.
It is essential to keep the cell density consistent for each transfection experiment.
The cells should be between 50-70% confluent at the time of transfection,
although this may need to be optimized for each cell type.
4.
The most important aspect of successful transfection is the careful optimization
of transfection conditions for each cell type. Several factors may influence the
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