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Documentation
Introduction
Microinjection or micromanipulation is the transfer of exogenous materials into or out of the cells by physically penetrating the plasma membrane with a glass micropipette or needle. The exogenous material might include proteins, sperms, peptides, cDNA, large non-diffusible drug molecules, and RNA injected into desired locations. Microinjection experiments were initiated by a bacteriologist, Dr. Marshal Barber, in 1902, and the first successful micromanipulation was performed in 1904. Microinjection system comprises three basic functions:
- Target cell imaging and micropipette
- Micropipette positioning
- Micropipette pressure control
Microinjection is a simple method requiring an inverted microscope, a micromanipulator device to control micropipette movement, and a microinjector. Injections are carried out under direct visual control using a microscope. The microscope provides enough working distance for injection needles, whereas a micromanipulator is used to position the needles. Extrusion of exogenous materials through the micropipette occurs by hydrostatic pressure. The microinjector builds positive pressure to inject materials and negative pressure to hold suspended cells. An injection chamber maintains the environment for cell survival. The small micropipette tips ensure accurate and precise delivery of the biological material. Conceptually, microinjection is a simple technique, yet it is difficult to apply (Xu, 2018).
Pronuclear microinjection is a technique used to create transgenic organisms by injecting genetic material into the nucleus of a fertilized oocyte. The pronuclear microinjection technique is commonly used to study the role of genes using animal models by cell transfection, where cells are delivered with foreign genetic substances to enhance or inhibit specific gene expression. Consequently, one can study their gene of interest. Cell transfection is of three types, i.e., biological, chemical, and physical. Microinjection is a physical method of producing transgenic organisms and has the greatest transfection efficiency among all three methods. With the advancements in technology, automated and robotic microinjection systems have been developed to inject many embryos per minute.
Since it is a physical delivery method, the only independent variable in these well-controlled experiments is the injected substance. The effects produced by the injected substance are isolated and largely amplified.
Protocol
Preparation
- Perform in vitro transcription and purification of the transgene using any commercially available kit.
- Prepare 10µl aliquots of the filtered transgene in microfuge tubes and store them at -80oC.
- Thaw one or two transgene stock tubes on the day of microinjection and make serial dilutions with concentrations 1ng/µl to 5ng/µl. Each dilution must be 20µl so that it is sufficient for the microinjection procedure.
- Prepare embryo handling and transferring assembly. For this, take drawn-out Pasteur’s pipette and hold the pipette’s large end with your fingers, hold the smaller end with the forceps, and soften this end over a Bunsen burner. Move the pipette out of the flame and pull in opposite directions.
- Score the glass 4cm away from the shoulder using a diamond pencil, thus breaking the pipette into two pieces. The larger piece will be relatively smooth, and you can use it as a ‘handling pipette’ for ova collection and transfer.
- A smaller pipette can be used as a part of embryo transfer assembly. Mount it on the pipette holder, align its tip using heat, polish it until the tip diameter reaches 150µm, and sterilize it under UV in a laminar flow hood. These tips are prepared in batches for embryo transfer and then stored in a petri dish until used.
- Insert the pipette holder into one end of the plastic tubing and attach a mouthpiece to the other. Insert a syringe filter somewhere inline.
- Perform vasectomies on 15 male mice using the proper surgical procedure and collect ova from the female mice. Rinse the ova in CZB and then place the ova in CZB micro drops such that each drop contains 100 ova.
- Incubate the dish at 37oC, in 5% carbon dioxide, at least one hour before pronuclear microinjection.
- Mate the females with vasectomized males to get pseudopregnant females a night before embryo transfer by housing at least ten females with one male.
- Keep checking for vaginal plugs. If the females are not used, they will continue their regular estrous cycle within two weeks.
Microinjection Procedure
- Place the DNA aliquot on ice and insert the blunt end of the injection pipette into the DNA solution. The pipette will be backloaded by capillary action via internal filament. Look for a bubble at the pipette’s shoulder. If the bubble does not appear, discard the pipette and use a new one.
- Retrieve the ova from the CZB microdrop, rinse them once with CZB/H, and place them in the CZB/H drop in a microinjector chamber.
- Turn on the microinjection and nitrogen gas supply and set the injector balance to continuous pressure of 0.3psi so that the CZB/H medium is not drawn back into the pipette.
- Work under an inverted microscope at 100X magnification and lower the holding pipette into a CZB/H droplet.
- Focus the pronuclei by adjusting the microscope at 200X magnification and separate good-quality embryos from poor ones using a holding pipette.
- Now at 400X magnification, push the injection pipette against the holding pipette to break the tip to create an opening. Make sure that the opening is not too large, otherwise replace the pipette.
- At the same magnification, use a holding pipette to capture the embryo and bring its pronuclei to the center or far side of the embryo. Focus via microfocus knob.
- Position the injection pipette tip at 3 o’clock and align it along the midline with the embryo so that its tip is next to the pronucleus. The mid-plane of the pronucleus and pipette must be in focus.
- Insert the injection pipette into the pronucleus, thus injecting the embryo. Once the pipette tip is inserted into the pronucleus, push the INJ button on the microinjector or step on the foot pedal to extrude the DNA solution. At this step, a slight expansion of the pronucleus ensures the right delivery.
- Gently and quickly, withdraw the pipette out of the pronucleus.
- Move the injected embryo to a different place within the drop to separate injected embryos from the non-injected ones.
- Repeat steps 7 to 11 to transfect all the embryos and transfer them to a fresh CZB/H dish.
- Wash the surviving transgenic embryos once in a dish of CZB medium and place them in groups of 25 into equilibrated CZB drops under mineral oil. The number of embryos in each group depends on the number of available recipients, but each group of embryos constitutes a cohort for transfer into a recipient.
- Incubate the embryos for half an hour in humidified 37oC temperature, in the presence of 5% carbon dioxide. This duration is sufficient to assess the surviving embryos that can be transferred.
- In surviving embryos, the plasma membrane will be drawn from zona pellucida.
- Transfer these transfected embryos to the pseudopregnant females following a proper surgical procedure.
Applications
The microinjection technique is extensively used to inject human embryos and mitotic cells. It can be successfully used to study primary cultured neurons that are difficult to transfect. It can also successfully deliver proteins (like recombinant caspases and neutralizing antibodies) and cDNA constructs into human neuronal cytosol. RNA interference transduction, IVF (in vitro fertilization), and transgenic animal production can also be achieved by microinjection.
Production of transgenic goats
Gavin et al. (2017) produced transgenic goats by engineering the desired DNA into the animal genome. They stated that these transgenic goats could be used to produce recombinant human protein therapeutics in their milk. To experiment, they acquired goats and ensured that donors and recipients were synchronized and superovulated. Both donor and recipient were injected with suitable hormones from time to time unless the implanted embryo was surgically removed and collected on day 14. They replaced the caprine ß-Casein coding region with the coding region of the gene of interest to prepare transgene (16.5-22kb in size). Moreover, for monoclonal antibodies, they co-injected DNA preparations with equal quantities of each construct that carried transgenes encoding heavy and light immunoglobulin chains. For microinjection, the researchers took a holding pipette and an injection pipette and attached them to individual manual syringe pipettors via polythene tubing infused with paraffin oil. Then, they loaded a glass dish with buffered embryo-holding media and a drop of DNA dilution, which was overlaid with oil. They evaluated the embryos for the presence of good quality pronuclei under an inverted microscope. They then injected the pronuclei (of single-cell and occasionally, two-celled embryos) with 4-8pL of DNA dilution using a microinjector. The microinjection caused the swelling of the pronuclei. The injected embryos were incubated at 90% humidity, 37oC, and 5% carbon dioxide for 2 to 4 hours. The viable embryos were surgically transferred to appropriate recipients. The animals were monitored regularly and allowed to give birth on the 150th day. Out of 18,120 microinjected embryos, 14.060 survived, with a survival rate of 77.6%. Out of 2594 offspring generated, 109 were transgenic.
CRISPR/Cas9 Microinjection for Knocking Genes
Vilarino et al. (2017) studied in vitro organogenesis by knocking out the PDX1 (pancreatic and duodenal homeobox protein 1) gene, which is critical for pancreatic development by microinjecting CRISPR/Cas9 in sheep oocytes. Following this, the researchers assessed whether PDX1 knockout would alter pancreatogenesis in sheep by microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte injection increased the number of biallelic mutations, reduced lysis, and showed a similar degree of mosaicism compared to zygote microinjection. They completely disrupted the PDX1 gene by using a dual sgRNA system. They concluded that PDX1-/- fetus lacked pancreas, and this mutation creates vacant space for interspecies organ generation.
Strengths and Limitations
Microinjection has manifold advantages over other mechanical delivery methods like electroporation, carrier peptide delivery, infection, or transfection. Microinjection delivers a precise volume of the injected material, at a precise time, into the cytosol or nucleus, which is difficult to control in many other methods. The researchers can readily identify the injected cells by co-injecting an impermeable marker dye or protein conjugated to a florescence. Additionally, the microinjection technique requires less protein preparation that can be beneficial while working with expensive recombinant proteins, less abundant proteins, or synthesized peptides. As compared to chemical transfection, or viral infection, this technique is relatively less stressful to cells. It also reduces the background resulting from cell death which is often observed with chemical or liposome-mediated transfections. Furthermore, the experimenter can add more than one construct in a single culture dish, using uninfected cells as controls, making the results more compatible. The theoretical efficiency of these procedures is 100%.
However, this technique has a few limitations as well. First, the microinjection technique is a ‘single-cell technique.’ It is rarely used for transducing a large number of cells and producing overly expressed cells for immunoprecipitation, bioluminescence, western blot, or purification. Common readouts of microinjection include immunofluorescence, cell death detection (in situ), translocation, and PCR. Furthermore, it is the most powerful technique to deliver nuclear and cytosolic proteins but fails to deliver membrane proteins, neurotransmitter receptors, or ion channels; however, one can achieve this by injecting cDNAs of these proteins. Second, injection is the physical humiliation of the cell. Therefore, vehicle control is requisite to exclude the possibility that injection alters cell viability. Third, positive controls and immunohistochemical staining is required to detect injected substances. Fourth, cell division constantly dilutes the injected substances in dividing cell lines, so long incubation after injection is not recommended.
Summary
- Microinjection is the transfer of exogenous materials into or out of the cells by physically penetrating the plasma membrane with a glass micropipette or needle.
- Microinjection is a simple method requiring an inverted microscope, a light source, a micromanipulator device to control micropipette movement, and a microinjector.
- Pronuclear microinjection is a technique used to create transgenic organisms by injecting genetic material into the nucleus of a fertilized oocyte.
- The microinjection technique is extensively used to inject human embryos, and mitotic and neuronal cells, for in vitro fertilization, organogenesis, and transgenic animal production.
References
- Liu, C., & Du, Y. (Eds.). (2019). Microinjection: Methods and Protocols. Springer New York.
- Vilarino, M., Rashid, S. T., Suchy, F. P., McNabb, B. R., van der Meulen, T., Fine, E. J., … & Ross, P. J. (2017). CRISPR/Cas9 Microinjection in Oocytes Disables Pancreas Development in Sheep. Scientific reports, 7(1), 17472.
- Xu, W. (2019). Microinjection and micromanipulation: a historical perspective. In Microinjection (pp. 1-16). Humana Press, New York, NY.
- Gavin, W., Blash S., Buzzel, N., Pollock, D., Chen, L., Hawkins, N., Howe, J., Miner, K., Pollock, J., Porter, C., Schofield, M., Echelard, Y., & Meade, H. (2017). Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995-2012). Transgenic Research, 27(1), (pp. 115-122).
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