The Microinjection System to Suit Any Application

Single-cell microinjection represents an innovative way to integrate exogenous material into cells and to extract and transmit cellular components between cells. The effectiveness of any microinjection system depends on the success of three processes; imaging of the target cell, placement of the micropipette, and pressure control within the micropipette.

XenoWorks™, developed by Sutter Instrument, is a state-of-the-art microinjection device which can satisfy the requirements needed for any high-performance microinjection and micromanipulation application.

Introduction to Microinjection

Workstation

In order to produce accurate and reliable outcomes, microinjection must be carried out in a suitable environment. Typically, this means a quiet location away from any possible causes of vibration, such as slamming doors or centrifuges, with a stable temperature, minimal draft and no excessively bright light.

Specially designed anti-vibration tables are a good solution for settings where vibration is still detected even after the necessary precautions have been implemented. These can either be created at home or are commercially available to buy.

Microscope

Inverted microscopes are generally used for microinjection applications. The working distance of the condenser, measured from the lowest part of the condenser to the stage, is very important. A long or ultra-long working distance enables the micromanipulator access to head stages with unhindered movement.

Stage attachments, like moving stages, specimen holders and heating/warming inserts, are potential sources of conflict with micromanipulator motors and so should be positioned carefully. The addition of a camera or TV monitor should also be taken into consideration as possible obstruction, however their inclusion may essential depending on what application the system is required for (e.g. teaching purposes).  

Micromanipulators

The micromanipulator – also known as the micropositioning device – must be firmly mounted to the microscope using a suitable adapter which can be obtained separately. The stability of the micromanipulator positioning is extremely vital and actually governs the success of the entire application. Good stability can be achieved by mounting the micromanipulator on a large footprint, with minimal overhang between the microscope mounting and the pipette holder clamp.

The micromanipulator is used to place the micropipette in certain immediacy to the target tissue. Some micromanipulators, such as the XenoWorks micromanipulator, are electronic. Such systems are highly advantageous as the software can automatically sense the position of the micropipette and is therefore able to move to any pre-determined spatial coordinate within the system’s range, achieving maximal accuracy even with the smallest of movements.

Microinjector

The microinjector maintains the pressure level inside the micropipette. No one pressure is ideal for every application – many different pressures may be needed depending upon the specific system use. For example, microinjection of fluids (like nucleic acid solutions or dyes and drug compounds) requires a lot of pressure to force the solution out of the very small tip, which usually has a diameter of less than one micron.

At any point when injection pressure is not being applied, a continuous positive “’base’ pressure should be applied to the tip. This prevents any of the medium from being drawn from the injection chamber up into the micropipette, thus diluting the solution. In Sutter’s XenoWorks, injection and base pressures in the digital microinjector can be controlled independently.

In addition to variable pressure, this injection system can also be set to distribute pressure pulses at specifically defined times, typically in the order of tens of milliseconds. This means that highly precise injections can be carried out from one target to the next with the simple press of a switch. Furthermore, any required pressure can be maintained continuously with XenoWorks by depressing the footswitch control.

Cellular Manipulation and Reconstruction

Relative to those used for direct pressure microinjection, micropipettes with a much wider tip are required for transfer and insertion of cellular components. A significantly narrower range of positive and negative pressures must be used for this process, and with this, more control must be exerted over the pressures applied. These limited pressure ranges are typically controlled using a micropipette holder linked by narrow-gauge tubing to a syringe whose plunger is connected to a micrometer leadscrew.

Turning the screw consequently changes the volume of the syringe, and hence enables precise pressure control in the micropipette. More modern systems utilize a hydraulic fluid instead of air in the syringe and tubing, creating an even superior level of control.

The XenoWorks analog microinjector an example of an instrument which uses this approach. This makes it ideal for use in a variety of complex applications, such as embryo holding and embryonic stem cell transfer. In addition, its ability to provide high pressure for solution injection makes the XenoWorks beneficial for use in transgenic animal production.

Microinjection Applications

1. Zygote Pronuclear DNA Microinjection

The microinjection of DNA into the pronucleus of a fertilized mammalian egg has become a frequently used approach to produce transgenic offspring. Although pronuclear microinjection was initially carried out in the mouse, several different transgenic animals have now been made using this method. As the micropipette used for injection is sharp, relatively high pressure (> 3000 hPa) is needed to transfer the DNA solution.

This application requires the use of two micromanipulators; one to hold the zygote and the other to inject the DNA. A subtle negative pressure is applied on the holding side, whilst rhythms of high pressure enable injection of the DNA solution into the pronucleus. Due to its concurrent holding and high-pressure injecting abilities, the XenoWorks digital microinjector is perfect for this application.

Suggested system configuration:

1 x XenoWorks Micromanipulator (Right) BRMR
1 x XenoWorks Micromanipulator (Left) BRML
1 x XenoWorks Digital microinjector BRE110/BRE220
2 x XenoWorks Microscope adapter BR-xxx

2. Embryonic stem cell transfer into blastocyst

Introducing genetically altered embryonic stem cells into the cavity of a blastocyst causes the stem cells to transfer genetic material to the embryo. The resulting animal is engineered with a combination of both genotypes. Two micromanipulators are needed for this process; one for holding the blastocyst and one for moving the cells. Both functions require mild positive and negative pressure. Although the digital microinjector is ideal for this, two analog microinjectors can be used as an alternative option.

Suggested system configuration:

1 x XenoWorks Micromanipulator (Right) BRMR
1 x XenoWorks Micromanipulator (Left) BRML
1 x XenoWorks Digital microinjector BRE110/BRE220

OR:

2 x XenoWorks Analog microinjector BRI
2 x XenoWorks Microscope adapter BR-xxx

3. Somatic Cell Nuclear Transfer

Genetically identical copies of an animal can be achieved with enucleation of an oocyte followed by the transfer of a somatic cell. Two micromanipulators are typically needed for this; one for holding the oocyte and the other for the enucleating and injecting processes. Each micromanipulator attaches to a solitary micropipette holder with a microinjector connected. Mild positive and negative pressure are required for oocyte holding, enucleation and somatic cell transplantation, of which analog microinjectors are therefore appropriate.

Suggested system configuration:

1 x XenoWorks Micromanipulator (Right) BRMR
1 x XenoWorks Micromanipulator (Left) BRML
2 x XenoWorks Analog microinjector BRI
2 x XenoWorks Microscope adapter BR-xxx

4. Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is a vital process used for veterinary fertilization in-vitro, which can aid the conservation of rare or endangered species. Less commonly, ICSI is used as a gene transfer procedure whereby sperm are co-injected with exogenous DNA.

Two micromanipulators are used for this; one for oocyte holding and the other for sperm aspiration and injection. As mild positive and negative pressures are needed for the intricate jobs of oocyte holding and sperm injection, an ICSI workstation ought to be configured with two analog microinjectors.

Suggested system configuration:

1 x XenoWorks Micromanipulator (Right) BRMR
1 x XenoWorks Micromanipulator (Left) BRML
2 x XenoWorks Analog microinjector BRI
2 x XenoWorks Microscope adapter BR-xxx

5. Piezo-assisted ICSI T

This is new approach is developed to aid conception in animals when standard ICSI has previously failed. It can also be used for gene transfer, whereby sperm are covered in exogenous DNA and subsequently injected into oocytes. The microinjection workstation required for this is similar to that used for standard ICSI but necessitates a piezo impact drive connected to the injecting micropipette holder.

In this approach, the micropipette vibrates and drills into the oocyte. The small amplitude yet high frequency of this vibration means a mechanically stable micromanipulator must be used. Good stability will ensure efficient energy transfer from the piezo impact drive to the micropipette tip.

Most piezo-assisted microinjection protocols require a bead of mercury to stabilize the injecting micropipette - mercury should not be used in combination with a digital microinjector, but the analog microinjector, on the other hand, is suitable.

Suggested system configuration:

1 x XenoWorks Micromanipulator (Right) BRMR
1 x XenoWorks Micromanipulator (Left) BRML
2 x XenoWorks Analog microinjector BRI
1 x PMM-150FU piezo impact drive with XenoWorks adapter
2 x XenoWorks Microscope adapter BR-xxx

6. Microinjection of Cultured, Adherent Cells

Cultured cell lines can be microinjected whilst still attached to a Petri dish. The best way to view this procedure is using phase-contrast optics with a micromanipulator and a high-pressure microinjection channel. This process involved bringing down the tip of a sharp micropipette onto a single cell before a pulse of high pressure is generated. The high-pressure ability of the digital microinjector along with the smooth control of the XenoWorks micromanipulator are both ideal for this application.

Suggested system configuration:

1 x XenoWorks Micromanipulator (Right) BRMR
1 x XenoWorks Digital microinjector BRE110/BRE220
1 x XenoWorks Microscope adapter BR-xxx

References

  1. Hiramoto, Y. (1984). Micromanipulation. Cell Struct. Function 9 Suppl. s139–s144.
  2. Kishimoto, T. (1986). Microinjection and Cytoplasmic Transfer in Starfish Oocytes. In: “Methods in Cell Biology”, Vol 27 (T.E. Schroeder ed.) 379–394. Academic Press.
  3. Gordon, J.W. et. al. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Nat. Acad. Sci. 77: 7380–7384.
  4. Hogan, B. et al. (1994) “Manipulating the Mouse Embryo – A Laboratory Manual”, 2nd edition. Cold Spring Harbor Laboratory Press.
  5. McGrath, J. and D. Solter (1983a). Nuclear transplantation in mouse embryos. J. Exp. Zool. 228: 355–362.
  6. Campbell, K.H.S. (1994). Biol. Reprod. 50:1385–1393.
  7. Meng, L. and D. P. Wolf (1997). Sperm-induced oocyte activation in the rhesus monkey: nuclear and cytoplasmic changes following intracytoplasmic sperm injection. Human Reproduction 12: 1062–1068.
  8. Perry, A.C.F. et al. (1999). Mammalian transgenesis by intracytoplasmic sperm injection. Science 284:1180–1183.
  9. Kimura, Y and R. Yanagimachi (1995). Intracytoplasmic sperm injection in the mouse. Biol. Reproduction. 52: 709–720.
  10. Graessmann, M and A. Graessmann (1983). Microinjection of tissue culture cells. Methods Enzymol. 101: 482–492.
  11. Proctor, G.N. (1992). Microinjection of DNA into mammalian cell in culture: Theory and practice. Methods Mol. Cell. Biol. 3, 209–231.

Sutter Instrument

For over 45 years, Sutter Instrument has been a recognized global leader in the design, and manufacture of specialized biomedical research instrumentation essential to biomedical research and emerging analytical techniques that utilize sub-micron probes and pipettes.

With distributors in over 38 nations outside the United States, our strong customer orientation, technical expertise and commitment to quality, has helped us develop an exceptionally loyal customer base that offer insights to advancing technologies and exhibit significant repeat purchasing patterns.

The unique synergy between our product groups (microprobe fabrication, micromanipulation, imaging, and micromanipulation) affords the company an ideal position for expansion and growth, especially in the high-growth fields of neuroscience, high-speed imaging, genomics, optogenetics, drug discovery and stem-cell research.  


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Last updated: Apr 1, 2019 at 4:07 AM

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