Application No. 2Sample
Preparation for Microinjection
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General Comments
Microinjection is the loading or transfer of a dissolved substance into a living cell. The
microscopic tip of the glass microcapillary has an inner diameter between 0.2 and 1 µm [1]. This capillary is back loaded with the substance to be
transferred into the cells cultured for microinjection. Typical substances include
purified antibodies, DNA, RNA, peptides, or oligonucleotides. |
To visualize and evaluate the success of a
microinjection experiment these substances are typically mixed with dyes or labeled with
fluorescent markers such as flourescein or rhodamine. After the capillary has pierced the
cell, a certain amount of transfer substance (approximately 10% of the cell volume) is
transferred from the capillary into the cell due to pressure exerted on the capillary via
the microinjector [2]. Because the diameter of the capillary
is very small, particles in the injection solution can quickly result in blockage of the
capillary. Therefore, exchange of the capillary is a common practice during microinjection
experiments and the frequency of the capillary exchange depends on the concentration and
clarity of substance injected.
To maximize the number of injections which can be performed with a
single capillary, the injection substance should be centrifuged for 1015 minutes at
a minimum of 10,000 x g before back loading the capillary. As the cross section of
the capillary tip is small and very little liquid would be found there, it dries quickly
and creates a blockage. Therefore, the filled capillary should be inserted into the
capillary holder and immediately lowered into the medium to avoid blockage due to drying.
The capillary should always remain in the medium especially during long experiments when
large numbers of cells are injected.
In order that the open petri dish has a sufficient pH stability
under the microscope, a carbonate-free medium must be used (e.g., HEPES, pH
7.4 buffered, 10 mmol/l). A 3 cm dish should be filled with at least 2 ml medium.
Cultivation can still take place in the usual medium containing carbonate.
The speed and efficiency of automated systems allows biochemical
analysis of microinjected cells. Between 500 and 1000 labeled cells are sufficient to
analyze labeled proteins by gel electrophoresis.
Cultivation and Microinjection
Preparation [3]
- Plate 250 cells in 5 µl droplets in the center of a glass coverslip
(10 x 10 mm).
- Place coverslips into a humid chamber and incubate at 37°C until
cells attach to the glass (usually takes 68 hr).
- Transfer coverslips into 35 mm petri dishes containing 2 ml of
culture medium and let cells grow for 2 days at 37°C. After this time, 500 to 1000 cells
will usually be in the center of the coverslip.
- Microinject all cells on the coverslip.
- Proceed with biochemical analysis (depends on the particular
experiment).
Note: For time series experiments, the cells may be plated directly
on to Eppendorf® CELLocate gridded cover slips placed in the
center of a glass slide. Thereby individual cells (55 µm) or cell groups (175 µm) can be
located easily after microinjection. Eppendorf Micromanipulator 5179 and FemtoJet
can be used with Femtotips and Femtotips II precision microcapillaries to efficiently
perform cytoplasmic and nuclear microinjection on a wide range of adherent cells.
Proteins, Antibodies
Purification [4] Purification methods which result in the
highest activity of the particular proteins or antibodies should be used. For peptide
antibodies raised in rabbits, affinity purification is recommended. The concentration of
the material injected should be 10 to 20 times higher than that required for the optimum
in vitro activity, because the sample is diluted 10 to 20 times when injected into the
cells.
Storage Purified antibodies must be stored in the concen-tration in
which they arise. Shock freeze small aliquots of 510 µl in liquid nitrogen. Store
at maximum temp. of 20°C (80°C is even better). Azide should not be used.
Refrain from repeated freezing and defrosting as antibodies lose
activity and start to agglutinate leading to blockages of the injection capillaries.
Defrosting should be performed as quickly and gently as possible.
Before loading the capillary, centrifuge material for 15 minutes at
4°C (10,000 x g). Chill supernatant or load directly into capillary.
DNA [5]
Purification The highest expression level of microinjected plasmid DNA is achieved when
the DNA has been purified by CsCl ethidium bromide gradient centrifugation in accordance
with the Maniatis protocol [6].
Dissolve DNA in bidist. water after purification.
Buffers, e.g., PBs, are not recommended as this often leads
to blockages of the capillaries.
DNA concentrations of 20 µg/ml to 200 µg/ml for plasmid DNA
injection has been recommended [1].
Storage Store at 20°C in small aliquots of 510 µl.
Defrosting should be performed as quickly and gently as possible.
Before loading the capillary, centrifuge material for 15 minutes at
4°C (10,000 x g). Chill supernatant or load directly into capillary.
Repeated centrifugation does not cause damage but is not necessary
for each capillary filling when chilled with ice and used within an hour.
RNA Purification
Any standard protocol is suitable for purifying RNA solutions [6].
As for DNA, RNA is dissolved in bidist. water after purification.
Concentrations of 12 µg/ml should be used for mRNA and up to
10 µg/ml for total RNA.
Storage Store at 80°C in small aliquots of 510 µl.
For longer periods, it is advisable to dissolve and store the
cleaned RNA in alcohol instead of water.
Defrosting should be performed as quickly and gently as possible.
Before loading the capillary, centrifuge material for min. 15
minutes at 4°C (10,000 x g). Chill supernatant or load directly into capillary.
Only centrifuge and use each aliquot once.
Dyes, fluorescent injection markers [1]
Successful fluorescent injection markers used to identify and follow injected cells are:
fluorescence labeled dextrans, antibodies, bovine serum albumin.
Dextran marked e.g. with rhodamine of a concentration of 2 µg/ml
can be detected for up to 48 hours in the cell.
Fluorescein fades with time and results in harmful radicals,
therefore markers should preferably be labeled with rhodamine.
Storage Store at 80°C in small aliquots of 510 µl.
Many solutions are sensitive to light. Thus, exposure to direct
light should be avoided.
To avoid blocking of the capillary during microinjection, solutions
containing the markers should be filtered with a syringe filter (pore size 0.2 µm)
whenever possible. Also, before loading the capillary, the sample should be centrifuged
for 15 minutes at 4°C (10,000 x g).
Peptides
Please refer to the current literature for information on the cleaning and use of
peptides.
A concentration of at least 510 µg/ml should be used for
injection, because some peptides rapidly degrade in the cells after injection.
Oligonucleotides
Purification The purification of oligonucleotide solutions is very important. Cleaning
with gel or HPLC is recommended.
Like DNA, oligonucleotides are dissolved in bidist. water after
purification.
A concentration of 12 µg/ml should be used for injection of
antisense oligonucleotides with 1020 bases.
Note: Injected oligos accumulate easily in the nucleus.
Literature
[1] Pepperkok, R., Schneider, C., Philipson, L., and Ansorge, W.
(1988). Single cell assay with an automated capillary microinjection system. Exp.Cell Res.
178, 369-376.
[2] Pepperkok, R., Scheel, J., Horstmann, H.,Hauri, H.P., Griffiths, G., and Kreis,T. E.
(1993b). ß-COP is essential for biosynthetic membrane transport from the endplasmic
reticulium to the Golgi complex in vivo. Cell 74, 71-82.
[3] Pepperkok, R., Saffrich, R., and Ansorge, W. (1994). Computer-Automated Capillary
Microinjection of Macromolecules into Living Cells. Cell Biology: A Laboratory Handbook,
CSH Laboratory Press
[4] Antibodies: A laboratory manual (1988). Harlow, E. and Lane, D. ed., CSH Laboratory
Press.
[5] Proctor, G.N. (1992). Microinjection of DNA into mammalian cell in culture: Theory and
practice. Methods Mol. Cell. Biol. 3, 209-231.
[6] Sambrook, Fritsch, Maniatis (1989). Molecular Cloning: A laboratory manual. CSH
Laboratory Press.
We thank Dr. Rainer Pepperkok, Université
de Genéve, Département de Biologie Cellulaire Sciences III, CH 1211 Genève 4, for
providing us with data. |