Download REVERSE GENETICS: USING RNAi TO MAKE PROTEIN KNOCK

REVERSE GENETICS: USING RNAi TO MAKE PROTEIN KNOCK-DOWNS
Developmental Biology Lab – Fall 2004
Using reverse genetics, one first identifies a gene of interest, and then determines what defect, if
any, results when the corresponding protein is missing. This approach may be used to
investigate whether a particular protein performs the same functions in one organism as a
homologous protein (one with a similar sequence) does in another organism. Using a simple
organism like C. elegans, one might investigate whether a C. elegans protein that is homologous
to a mammalian protein has a related function. If so, C. elegans might then be used as a model
system to study the normal function of the protein, the regulation of the protein, and molecular
targets of its action. If the absence or mutation of the mammalian version of the protein leads to
a disease, studying its C. elegans homolog might further our understanding of the molecular
basis of the disease and could elucidate possible treatments.
There are several different strategies for eliminating or severely depleting the expression of a
particular protein, which are referred to as “knock-out” or “knock-down” strategies, respectively.
C. elegans researchers, among others, employ a technique called RNA mediated interference, or
RNAi. The central dogma of molecular biology is that DNA is transcribed into mRNA, and
mRNA is translated into protein. Therefore, one would assume that more of a certain mRNA
would result in more of that protein. This is true to a certain extent. What we now know is that
if you introduce an extreme amount of a specific dsRNA into a worm, that dsRNA gets
processed and leads to the degradation of the corresponding endogenous mRNA. This technique
might not completely eliminate the corresponding protein, but it significantly diminishes the
protein levels, thereby resulting in a phenotype that mimics a strong loss-of-function mutant.
Remarkably, the mRNA can be introduced either by injection, soaking the animals in a solution
of the mRNA, or even by feeding it to the animals on a plate!
Each group will be responsible for performing the RNAi feeding protocol on one C. elegans
gene. Due to our limited time together, the experiment will be spread out over the next four lab
periods. Some of the preparatory tasks will be performed for you.
SEE TABLE ON REVERSE SIDE.
DATES:
TASKS:
Th 12/2; F 12/3
- Make M9-lactose plates for RNAi feeding
protocol
- Observe worms by DIC and learn basic worm
anatomy
- Bleach N2 hermaphrodites and allow embryos to
hatch in M9
- Start overnight bacterial cultures from newly
transformed plates of L4440 alone or L4440 with
cDNA of interest
- Seed M9-lactose plates with O.N. culture (L4440
alone or L4440 w/clone); allow to dry
- Place L1 larvae on seeded plates
- Observe N2 hermaphrodites with
stereomicroscopes
- Analyze data from RNAi feeding experiment
- Characterize phenotypes using stereomicroscopes
and DIC; document info
- Use computer databases to find info regarding
your gene/protein of interest (What type of protein?
Any conserved domains?)
- Use databases to determine whether humans have
a homologous sequence. If so, is the function
known?
M 12/6
T 12/7; W 12/8
Th 12/9; F 12/10
T 12/14; W 12/15
ACCOMPLISHED
BY:
BIO 408 students
Other
BIO 408 students
BIO 408 students
BIO 408 students
FINAL WRITTEN REPORT – 30 POINTS
Developmental Biology Lab – Fall 2004
The following questions or requests should be addressed in a written report. One report per lab
group of three should be submitted by 10 a.m. on Friday, December 17. The report should be
typed, double-spaced, and have one-inch margins on all sides.
The report grade will be based on the group’s ability to:
- address all requested points
- accurately and completely answer questions
- demonstrate overall good writing skills; organization, topic sentences, proper
grammar, correct spelling, and punctuation
1) What does “RNAi” stand for?
2) What is the purpose of RNAi? Why do researchers perform these kinds of experiments?
3) What is the cellular mechanism by which RNAi elicits its effects?
4) In your own words, briefly describe how the dsRNA gets generated and gets into the
worm.
5) What protein did you deplete by RNAi and what served as the control in the experiment?
6) What phenotype(s) did you observe in comparison to the control? DOCUMENT WITH
FIGURES.
7) Based on the observed phenotype, what organ system(s) might be affected?
8) What type of protein did you RNAi (i.e. a specific kinase, a cell-cycle protein, etc)?
9) Are there any conserved domains? If so, what are they and what functions are they
associated with?
10) What kind(s) of cellular activities is this type of protein involved in?
11) Is there a human ortholog (a human version) of this protein?
12) If so, what is its name and what type(s) of cellular activities is it involved in?
13) Based on the results of the bioinformatics and any primary literature resources that you
might have found, predict the molecular basis for the observed phenotype.