Xenopus Embryo Microinjection

Intuitive and ergonomic microinjections in Xenopus with the MK1 Micromanipulator from Singer.

Esther Pearl and Marko Horb

National Xenopus Resource, Marine Biological Laboratory, USA.

 

Microinjection is an important technique used by many Xenopus researchers to inject into embryos for loss-of-function 1 and gain-of-function studies 2,3, making transgenic 4,5,6 and knock out lines 7, as well as injecting into oocytes 8,9,10. Figures 1 and 2 show the right-handed MK1 (MK1-001) being used with a Singer pipette holder (INK-001) and glass capillary needle to inject mRNA into Xenopus embryos.

The Singer MK1 Micromanipulator allows us to microinject Xenopus oocytes and embryos using natural hand movements, as opposed to using knobs, thus it is very intuitive and your hand never has to leave the hand grip. The MK1 moves the needle in the same directions as the hand moves, only the movement is reduced by a 1:4 ratio. This allows fine control of the injection needle, enabling precise injections. The MK1 can be rotated so that the needle is parallel to the bench, or on an angle, without the need for an additional tilt mount.

More detailed view of the MK1 micromanipulator and pipette holder (INK-001) in use during microinjection.

 

Figure 1

An example using the right-handed MK1 (MK1-001) and the pipette holder (INK-001) to inject mRNA into Xenopus embryos. Asterisk indicates the bar that tilts to change the angle of the pipette holder.

 

The National Xenopus Resource (NXR), based in Woods Hole, Massachusetts, USA, is a resource center for Xenopus research (www.mbl.edu/xenopus). We offer custommade transgenic frog lines and knock out lines as well as maintaining existing frog lines. At the NXR we use the MK1 Micromanipulator for all picoinjection procedures.
The NXR creates transgenic frog lines, TALEN and CRISPR knock out lines, and performs oocyte host transfer (transferring oocytes that have been injected with mRNA into a host female to be laid and then fertilized) with the help of the MK1.

We routinely inject into Xenopus embryos and oocytes at various stages, including 1 cell, 2 cell, 8 cell and up to 32 cell stages. This allows us to target specific areas of the developing embryo if necessary. The benefit of using the MK1 is that it is much easier to control the area of the embryo we are injecting. Some of our injections are into the vegetal pole and use of the MK1 allows for precise injection into the vegetal hemisphere, which is the heavier side of a dejellied embryo, and thus on the bottom. The angle of the pipette holder can easily be adjusted at any time throughout the injection procedure, by tilting the bar where the pantograph meets the horizontal bar connecting to the stand (see asterisk in Figure 1).

Being able to use natural hand movements enables new users to adapt to and use the micromanipulator effectively very quickly, and has an ergonomic design reducing muscle fatigue which in turn increases efficiency enabling us to inject more embryos in a day. The NXR regularly injects hundreds, often thousands, of embryos a day; the MK1 enables us to perform efficiently.

More detailed view of the MK1 micromanipulator and pipette holder (INK-001) in use during microinjection.

Figure 2

More detailed view of the MK1 micromanipulator and pipette holder (INK-001) in use during microinjection.

 

 

 

REFERENCE

1. Pearl, E. J., Jarikji, Z., and Horb, M. E. (2011). Functional analysis of Rfx6 and mutant variants associated with neonatal diabetes. Developmental Biology. 351: 135-45.
2. Gurdon, J. B., Lane, C. D., Woodland, H. R., and Marbaix, G (1971). Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature. 233: 177-82.
3. Amaya, E. (2005) Xenomics. Genome Research. 15: 1683–1691
4. Kroll, K.L. and Amaya, E. (1996) Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development. 122: 3173–3183.
5. Ogino, H., McConnell, W.B. and Grainger, R.M. (2006) Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease. Mechanisms of Development. 123:103– 113.
6. Pan, F.C., Chen, Y., Loeber, J., Henningfeld, K. and Pieler, T. (2006) I-SceI meganucleasemediated transgenesis in Xenopus. Developmental Dynamics. 235: 247–252.
7. Lei, Y., Guo, X., Cao, Y., Deng, Y., Chen, X., Cheng, C. H., Dawid, I. B., Chen, Y., and Zhao, H. (2012). Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proceedings of the National Academy of Sciences. 109: 17484-9.
8. Laskey R. A., Gurdon, J. B., and Crawford, L. V. (1972). Translation of encephalomyocarditis viral RNA in Oocytes of Xenopus laevis. Proceedings of the National Academy of Sciences. 69: 3665-3669.
9. Olson, D. J., Hulstrand, A. M., and Houston, D. W. (2012). Maternal mRNA knock-down studies: antisense experiments using the host-transfer technique in Xenopus laevis and Xenopus tropicalis. Methods in Molecular Biology. 917: 167-82.
10. Munoz C., Pakladok, T., Almilaji, A., Elvira, B., Decher, N., Shumilina, E., and Lang, F. (2014). Up-regulation of Kir2.1 (KCNJ2) by the serum and glucocorticoid inducible SGK3. Cellular Physiology and Biochemistry. 33: 491-500.
 

 

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