Lab 4 (RNai and CRISPR/Cas9 to control gene expression) Flashcards
What are the objectives of this lab?
1) Understand the principles and applications of RNAi and CRISPR/Cas9 in gene expression control.
2) Prepare total cell extracts and quantify protein concentration using techniques from previous labs.
3) Perform Western Blotting to assess protein expression levels.
4) Analyse and interpret Western Blot data to determine the effectiveness of gene knockdown or knockout.
5) Quantify lipid droplets using immunofluorescence and microscopy.
What is the background for this lab? What is RNAi and CRISPR/Cas9?
- Lab 4 focuses on the powerful techniques of RNAi and CRISPR/Cas9 for manipulating gene expression. These technologies allow researchers to study gene function by either reducing gene expression (knockdown) using RNAi or completely removing the gene (knockout) using CRISPR/Cas9.
(1)
RNAi utilizes small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) to target and degrade specific mRNAs, effectively reducing protein production. While RNAi is a valuable tool, it doesn’t completely eliminate gene expression, which can sometimes lead to ambiguous results.
(2)
CRISPR/Cas9 provides a more precise and permanent method for gene editing. This system uses a guide RNA (sgRNA) to direct the Cas9 enzyme, a nuclease, to a specific location in the genome, where it creates a double-strand break. This break can then be repaired by the cell’s DNA repair mechanisms, often resulting in small insertions or deletions that disrupt the gene, effectively knocking it out.
What is the experiment summary?
In Lab 4, you will study the effects of RNAi and CRISPR/Cas9 on the expression of Adipose Triglyceride Lipase (ATGL), a key enzyme in lipid metabolism. ATGL is a 55 kDa protein that hydrolyses triglycerides to release fatty acids and diglycerides. It is highly expressed in adipose cells and plays a crucial role in lipolysis, the breakdown of lipids within cells.
(1)
Week 1: Prepare total cell extracts from unknown cell pellets (treated and control) and determine protein concentration using the Bradford assay, as practiced in Lab 1. Perform SDS-PAGE and Western Blotting to analyse ATGL protein expression levels in the samples.
(2)
Week 2: Analyse and interpret Western Blot data to identify the samples treated with shRNA (knockdown) and sgRNA (knockout) by comparing them to their respective controls. Quantify lipid droplets in fixed and stained cells using immunofluorescence microscopy and image analysis software to assess the effects of ATGL knockdown or knockout.
Cell Lysis and Protein Extraction
Cells are lysed using a buffer containing detergents (NP-40) to disrupt cell membranes and release proteins. Protease inhibitors are added to prevent protein degradation.
Bradford Assay
A colorimetric assay used to determine protein concentration based on the binding of Coomassie Brilliant Blue dye to proteins.
SDS-PAGE
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis is used to separate proteins based on their molecular weight. SDS denatures the proteins and gives them a uniform negative charge, allowing them to migrate through the gel towards the positive electrode. Smaller proteins migrate faster than larger proteins.
Western Blotting
A technique used to detect specific proteins in a complex mixture. Proteins separated by SDS-PAGE are transferred to a membrane, which is then incubated with antibodies that specifically bind to the protein of interest. A secondary antibody conjugated to an enzyme (horseradish peroxidase, HRP) is used to detect the primary antibody. The enzyme catalyses a reaction that produces a chemiluminescent signal, which can be visualised using a ChemiDoc imaging system.
Immunofluorescence Microscopy
A technique used to visualise the distribution of specific proteins or structures within cells. Cells are fixed and permeabilized, then incubated with antibodies that bind to the target protein. Fluorescent dyes (fluorophores) conjugated to the antibodies allow for visualisation of the target protein under a fluorescence microscope.
Image Analysis
Software, such as ImageJ, is used to analyse microscopy images. This software can quantify the fluorescence intensity of specific regions of interest (ROIs), allowing for the measurement of protein expression levels or the number and size of cellular structures.
Cell Lysis Buffer
Contains detergents (NP-40) to disrupt cell membranes, salts for osmotic balance, EDTA to chelate metal ions and inhibit metalloproteases, and protease inhibitors to prevent protein degradation.
Bradford Reagent
A dye that binds to proteins and changes colour, allowing for the measurement of protein concentration using a spectrophotometer.
SDS-PAGE Reagents
Includes acrylamide, bis-acrylamide (for gel formation), SDS (for protein denaturation), buffers for pH control, and TEMED and APS (for polymerisation).
Western Blotting Reagents
Includes transfer buffer, blocking buffer (to prevent non-specific antibody binding), primary antibody (specific to the target protein, ATGL), secondary antibody (conjugated to HRP), and chemiluminescent substrate (ECL) for detection.
Immunofluorescence Reagents
Includes fixative (formaldehyde), permeabilization solution (Triton-X 100), primary antibody (specific to lipid droplets), fluorescently labelled secondary antibody (BODIPY), and DAPI (a fluorescent dye that stains nuclei).
Describe the mechanisms of action of RNAi and CRISPR/Cas9 in controlling gene expression. What are the advantages and limitations of each technique?
(1) RNAi
- Mechanism: RNAi utilizes small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) that are complementary to the target mRNA. These small RNAs are incorporated into the RNA-induced silencing complex (RISC), which then binds to the target mRNA and cleaves it, leading to mRNA degradation and reduced protein production.
- Advantages: Relatively simple and cost-effective, can achieve significant knockdown of gene expression.
- Limitations: Transient effect (not permanent), potential for off-target effects (silencing of unintended genes).
(2) CRISPR/Cas9
- Mechanism: CRISPR/Cas9 uses a guide RNA (sgRNA) that is complementary to a specific DNA sequence in the target gene. The sgRNA guides the Cas9 enzyme, a nuclease, to the target site, where it creates a double-strand break in the DNA. This break is then repaired by the cell’s DNA repair mechanisms, often resulting in insertions or deletions (indels) that disrupt the gene, leading to a knockout.
- Advantages: Highly specific, permanent gene knockout, can target multiple genes simultaneously.
- Limitations: More complex and expensive than RNAi, potential for off-target effects (although these are being minimized with improved sgRNA design and Cas9 variants).
