Background to Project

Molecular Embyology Sign

A research project to study the differentiation of neuron cells in chick brains.
Overview
Over the summer 2013, I have been awarded a 4 week Nuffield Scholarship to work alongside researchers at the Institute of Biological and Biomedical Sciences (IBBS) at the University of Portsmouth on this project. The aim of the project is to work towards confirming the role of the gene Cellular Retinoic Acid Binding Protein 1(CRABP1) in neuron differentiation, in other words, the specialisation of a general purpose cell, or a stem cell, into one which acts as a neuron in the brain. This process of differentiation occurs whilst a chick embryo is developing, and is the beginning of the formation of the brain.

Not only is this an important strand of research, it is an exciting one where techniques including: cloning, working with plasmids (small pieces of circular DNA in bacteria) and DNA hybridisation (combining different strands of DNA) are commonly used.

Using these techniques, we hope to find further evidence that CRABP1 is in fact contributing to the process of differentiation of these neuron cells. If this is the case, a paper will be published to work towards establishing more details of its role. I will write up the project as it progresses and then report the findings in October in London.

Over the four weeks I will post updates on the progress of the work. I hope to describe the technical processes used and to convey the excitement of each stage as we progress through the research. A proportion of the work we will do will be microscopic but it is also very visual, so I will be posting images too. Here is my first one:

Where I will carry out my research work.

Where I will carry out my research work.

Background to the Project
Over the duration of my Nuffield Project, I will be primarily examining the expression of the gene CRABP1, Cellular Retinoic Acid Binding Protein 1. To identify when and where the gene CRABP1 is expressed in a developing embryo, we will be examining cells in the region of the chick embryo that will become the brain. A chick embryo has a number of stages and this work examines those between stages 9-12. These stages were proposed by Hamilton and Hamburger and define the stages of development of a chick.

The stages of development of a chick embryo. Source: www.summagallicana.it

The stages of development of a chick embryo. Source: http://www.summagallicana.it



How will we do this?

The genetic code of an organism is contained in its cells. At any one point, some genes may be activated and some suppressed because not all genes are required in every cell, for example a brain cell does not need a gene which is responsible for insulin production to be activated, whilst a pancreatic cell would. Those genes that are activated or “expressed” will be present in the messenger RNA molecules within the cell. These RNA molecules will contain a sequence of bases (A, U, C and G) which code for different genes.

An RNA molecule is very similar to DNA in structure, except it is single stranded. DNA can be visualised as a spiral ladder (a double helix), on which each rung is made up of two bases joined by hydrogen bonds (see diagram). RNA can be visualised like this, as a single sequence of bases or “half a ladder”.

We can insert into a cell a piece of RNA, known in this context as a probe, which contains the base sequence (in reverse order to make it complementary) for a specific gene, in our case CRABP1. This inserted RNA would bond with any messenger RNA within that cell that had the same sequence i.e. contained the same gene.

To identify where the RNA probe has bonded with messenger RNA in such an experiment we can attach a chemical to the probe which can act as a signal when we test for it. I will explain this process once I have experimented with it, but it basically is involved in staining the colour blue any of the cells that contain the gene we are looking for. We can then photograph the embryos under a microscope, and look for any blue cells. Using different genes and embryos at different stages, we can examine and compare when and where a gene is expressed and how this relates to other genes that are thought to be involved in the differentiation of the same types of cell.

This process can provide evidence for the presence and role of particular genes, in this case CRABP1 and can be the basis for further research into a gene.

For my Nuffield placement, I will research the gene CRABP1 and compare it to the genes Sox 2, Notch 1, C Mash 1, C Neuro M and C Neuro D, all believed to be involved in neuron differentiation in chick embryos, and so possibly in other species as well.

The idea that the outcome of these experiments is yet to be discovered makes this research very exciting. Over the next few weeks I will post a weekly blog with an update on the progress of the research and the various methods I will use.

The laboratory work
The lab work will involve:
• Making RNA probes to bind with the messenger RNA, which will using plasmids, making DNA templates, PCR and hybridisation, centrifugation and running on a gel

Cenrtifuge

Cenrtifuge

• Preparing and washing embryos, which will involve using microscopes, making up washing solutions in the chemical room and lots of pipetting!

The Chemical Room

The Chemical Room


The microscope

The microscope

• Staining by using an antibody, and an enzyme and its substrate
• Photographing the stained cells of the embryo under a microscope

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