Wednesday, 22 December 2010

Lab 1: Only the Nose Glows (The reality)

I’m back home for Christmas and all done with my first rotation. I’m going to start writing it all up tomorrow, 7000 words in 4 weeks. So it seemed like a good time to write a post about what I’ve actually been doing over the last 9 weeks.

I’ve been trying to find out what makes olfactory ensheathing cells (OECs) from neural crest stem cells (NCCs). This means I’ve been looking at which genes are active in these cells at different points in development. The information from an active gene is converted into a messenger molecule (mRNA) and this information is then used to construct a protein, which has a specific role within a cell. mRNA and proteins can be coloured using specific markers.

*WARNING* The following describes work with embryos (feel free to skip ahead)

See Embryo Work

Embryos are removed from their eggs at early stages. A chick takes approximately 21 days to develop fully. I have been working with embryos between day 3.5 and 10.5 of development. I have also done some grafting work with embryos at day 1.5. I take cells from embryos which make a protein in all their cells which is fluorescent green (GFP – Green Fluorescent Protein). This protein occurs naturally in certain jellyfish, and is often used in other animals by scientists. By transplanting neural crest cells from these green embryos into normal (non-green) embryos, I can see where all the cells migrate to later in development. So it is easy to see the OECs (which are green) on the nerve (which isn’t). This is how the lab demonstrated where OECs come from in the first place; by marking OECs, I can see which proteins are inside them.
Live embryos will continue to grow inside the egg after transplantation.

An embryo 2 days after transplantation.

The same embryo under blue light, showing the green transplanted cells.

The embryos are fixed, which makes them strong by linking everything inside the cells together. I then cast them into wax blocks allowing thin layers, one cell think, to be cut away and placed on microscope slides. I remove the wax once the cells are on the slides and can then stain them for the target molecules.

Cells were placed on slides in thin layers. I found which slides had OECs on them by looking for the olfactory system (nose) under a microscope and was then able to stain these slides for mRNA and proteins of interest.

In situ hybridisation (ISH) is used to locate mRNAs. RNA strands can bind to each other specifically in the same way that the two strands of DNA do in a helix. By making a short RNA probe, which binds to the mRNA I’m interested in, I can find which cells express it. The short probe is modified so that it also binds a protein that can react with certain chemicals to produce a colour. The probe is allowed to bind mRNA in the cells and then the colour is developed. The coloured cells can be easily seen with a microscope. The protein that I used is called alkaline phosphatase and it produced a purple colour. This process takes at least three days, although it can take up to a week for the colour to appear.

In Situ hybridisation stain for mRNA from the Sox10 gene. Marks all neural crest cells. Here showing the OECs on the olfactory nerve at day 5.5 of development. The nose (olfactory epithelium) is on the right and the brain (olfactory bulb) is on the left.
Immunohistochemistry (IHC) locates proteins. Your blood is filled with large proteins called antibodies, which attach to germs that invade your body and help destroy them protecting you from illness. It is possible to make antibodies that will specifically target almost any protein and several companies are based around doing just this. I used these antibodies, with an attached dye, to find proteins in the same way as the RNA probe finds mRNA. Usually I use a fluorescent dye; when exposed to one colour of light it will glow with a different colour. Fluorescence gives very clear images under a fluorescent microscope. Filters are used so that you only see the colour from the fluorescent stain. It takes about two days to stain for a protein.
Immunohistochemistry stain for nerve cells (on the same cells as the ISH above), using the TuJ1 antibody, which binds to a nerve specific protein, tubulin-beta.

All of these can also be used together on the same cells, allowing for comparison of lots of markers. On any one slide I can stain for up to three proteins and one mRNA. The three proteins use different fluorescent dyes, which glow red, green or blue. I am particularly looking for proteins which other cells from the neural crest are known to use during development. To know where the OECs are, some of the stains are for marker proteins (proteins that we already know are expressed in certain cells).

I now have a lot of work to do, going through all of the images to sort out what I’ve actually found and report it in a useful way. Other members of the lab (who have much more time than I did) will then have to repeat this work to confirm what I found.

1 comment:

Unknown said...

Nice explanation. Enjoy your holiday and please keep us up-to-date on the experiments. -- GeePawHill