Lab 6: Series 3-Reverse Genetics: Investigating Gene Function & Regulation Using RNAi
In the age of genome sequencing we now know, or can make educated guesses about, the location of every gene in an organism's genome; however, this does not give us any information about the function of the gene product (protein) in the organism. We can use reverse genetic analysis to help us solve this puzzle. There are several tools in the reverse genetics toolbox: directed mutation (point mutations or deletions), overexpression using transgenes, and gene silencing using knockout organisms or double stranded RNA (RNAi). Only RNAi and overexpression have been perfected in C. elegans. Scientists still have not found a way to do in vivo homologous recombination in worms.
We are going to use RNAi as our tool to investigate gene function via reverse genetics. C. elegans is the first animal in which the process of RNAi was discovered. A similar system was identified in plants years earlier but, curiously, that groundbreaking discovery was largely ignored by the scientific community until it was noticed in animal models. We now know that RNA regulation in cells is a fundamental method of regulating gene expression in organisms ranging from microscopic C. elegans to humans. Many labs are now working non-stop to develop treatments for many "incurable" human diseases using RNAi.
In this investigation for Series 3 that you will begin today, each pair of students will be given a strain of C. elegans that have been genetically manipulated to contain green fluorescent protein (GFP) under the control of the hsp-16.2 promoter. GFP is a 26.0 kDa protein comprised of 238 amino acids. When exposed to blue/ultraviolet light the protein emits bright green fluorescence. GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry for their discovery and development of the green fluorescent protein. This protein has been used in many different organisms ranging from bacteria and simple eukaryotes like S. cerevisiae the budding yeast, to zebrafish, cats and rabbits to examine protein localization and often as a biosensor. The DNA code for GFP is added to a gene of interest - usually on a plasmid - and then introduced into a cell. For mammals that cell is usually an egg or an early embryo. The DNA is then incorporated into the genome. When the gene of interest is turned and transcribed so is the code for GFP. The two genes are translated together and the resulting protein is the protein of interest with the GFP protein attached to it. When the organism is exposed to blue/ultraviolet light any cell or part of a cell where that protein is present will glow green.
The strain you will obtain today is called CL2070. In this strain the DNA code for GFP immediately follows the promoter for hsp-16.2. hsp-16.2 encodes a 16 kDa protein that is a member of the heat shock family of proteins. The expression of these proteins is upregulated during heat shock or environmental stress. HSP-16.2 protein is likely to function as a passive ligand temporarily preventing unfolded proteins from aggregating.
To Do Today
- Obtain a plate of CL2070 worms from your instructor.
- Examine the worms under the dissecting microscope. What is different about this strain of worms than any other you have seen thus far?
- Each pair should obtain 4 maintenance plates. Label 2 of them with CL2070 and 15°C and the other 2 plates with CL2070 and 23°C.
- Each pair should have a scalpel at their station. Flame sterilize the scalpel in your Bunsen burner flame - both sides. Let cool for 30 seconds.
- Cut 4 squares out of your CL2070 plate, about 1 cm on each side. Using the scalpel scoop out each square, one at a time, and flip it, worm side down, onto the plates you just labeled. Each plate should have one square of worms.
- Put your plates in the proper incubator to use next week.