Lecture 3 - basic principles and techniques (part 2) Flashcards
(33 cards)
What can single cell RNA-sequencing describe?
the molecular phenotype and define the cellular composition of complex tissues
What are the steps involved in single cell RNA-sequencing?
- Dissect intact tissue
- Prepare a dilute suspension of disaggregated single cells
- Sequence the transcriptome of each single cell separately
- Count transcript numbers for each gene in each cell
- Assign gene expression differences as being relatively high or relatively low for each cell
- Visualise and compare ‘Read Count Profiles’ (all genes) between individual cells.
- Cluster cells with similar Read Count Profiles into groups using a Statistical Method (Principal Component Analysis)
- Identify cell types
What does RNA-sequencing establish?
Allows analysis of which genes are upregulated and which genes are downregulated.
How can Single Cell RNA-seq (sc RNA-seq) be used in mutants?
Can be used to help understand the molecular and cellular basis of complex phenotypes
Can be used to test whether cell types have changes within a tissue
These are all techniques used to analyse gene expression.
What is a technique to analyse gene expression?
Single Cell RNA-sequencing
What are 2 techniques for analysing protein expression?
- Immunohistochemistry
- Visualisation of fluorescent fusion protein
What is immunohistochemistry?
Analysis of the spatio-temporal distribution of proteins in embryos with antibodies
Explain the process of immunohistochemistry
- Antibodies are made by injecting animals (e.g. rabbits) with the protein that you want to detect.
- Animal’s immune system recognises protein as foreign and produces antibodies that stimulate the immune system. Antibodies bind to small regions of proteins with very high specificity - called an epitope.
- Tagged antibodies have dyes or enzymes attached (conjugated) to them so that we can determine their location.
- Some conjugate the dyes that are fluorescent, allowing us to detect the location using specific wavelengths of light. Sometimes we use antibodies to exam the sub-cellular localization of the protein, other times we are testing where is the protein in the organism (which tissues).
FLUORESCENCE PROVIDES HIGH CONTRAST
What are 2 enzymes commonly used to conjugate?
- Alkaline phosphate - substrate turns blue
- Horseradish peroxidase - substrate turns brown
Why are enzymes used in inmmunohistochemistry?
Enzymes used as they amplify signal. Enzyme detection can enhance sensitivity.
We often make a 2-antibody sandwich, this amplifies the signal. This is because many secondary antibodies bind to each primary antibodies.
This is easier & cheaper as 2ndary antibodies are easily produced. This also leads to amplification, as 2nd antibodies recognise more than 1 epitope, leading to multiple copies of secondary antibodies.
Describe RNA localization of Bicoid in early drosophila embryo
- Tightly localized in anterior part of embryo. This is before cell has closed (cellularization).
- Bicoid protein is making a gradient across the whole anterior/posterior axis.
- This is more informative than mRNA. Once RNA is made into protein, the protein diffuses out.
Explain visualization of fluorescent fusion protein works
- Can use fluorescent fusion protein to view live samples.
- Variation of recorder lines. Fusion of GFP to gene.
- This allows GFP to be expressed after translation.
- GFP molecule is very compact (folds on itself) and doesn’t interfere with other proteins much.
What are the 2 types of Muller’s Morphs?
- Loss of function mutations
- Gain of function mutations
What are the different types of loss of function mutations?
- Amorphic
- Hypomorphic
- Antimorphic
What is the type of gain of function called?
Hypermorphic
What is an amorphic mutation?
Complete loss of function (null mutation/knock out). Most genes are haplosufficient in diploid organisms so these are usually recessive.
What is a hypomorphic mutation?
reduction of wild-type function. Usually recessive
What is an antimorphic mutation?
Competitive inhibitors - called dominant negative
What is a hypermorphic mutation?
Increased activity of the gene product - dominant
What is forward genetics?
Phenotype to gene
- seeks to identify a gene whose mutation caused a particular phenotype
What is forward genetics?
Gene to phenotype
- seeks to characterize the phenotype of particular mutated gene, by targeted mutagenesis
Describe the steps in forward genetics
- Randomly mutate the genome (chemicals like ENU or EMS)
- Look for interesting phenotype in the offspring
- Identify the gene that causes the defect.
Why are C. elegans, Drosophila and zebrafish the main animals used for FORWARD genetic screens?
As random mutagenesis affects the whole genome. This means that analysis of MANY mutagenized animals must occur, to find interesting phenotypes.
What are some possibilities for genetic screens?
- loss of certain cells or tissues
- disease-like phenotypes
- biochemical abnormalities
- loss of hearing or vision
- behaviour
- drug addiction
