DevBio Toolkit Flashcards
List the seven parts of the tool kit discussed in class
Lineage tracing, in situ, hybridization, immunohistochemistry, genetics, deep sequencing (RNA), transgenic organisms, CRISPR/Cas9-mediated gene editing
List the four different types of lineage tracing.
Direct observation, fate mapping using dyes, fate mapping with transgenic DNA, transgenic memory cassettes.
What is required for direct observation lineage tracing?
Organisms with unique cells that retain their uniqueness throughout development.
What are the two different types of fate mapping using dyes?
Vital staining and fluorescent dye.
Describe vital dye staining
Introduce dye into a group of cells early, see which cells are stained later in development
What is required for vital dye staining lineage tracing?
Being able to open the embryo or having a somewhat transparent embryo
What is a drawback of vital dye staining?
The dye dilute as cells divide
How is fluorescent dye different than vital dye staining?
Fluorescent die does dilute, but it is visible regardless of embryo transparency
Describe using genetic markers as cell lineage tracers
Transplant a cell from a similar organism to the organism of interest in its equivalent location
How is lineage tracing visualized through genetic markers?
Based on morphology or using antibodies
Why are genetic markers as so lineage tracer no longer common practice?
You need similar organisms, time intensive, not used in utero
Describe fate mapping with transgenic DNA.
Transgenic embryo produced in which every cell actively expresses GFP
Early in development group of cells is transplanted into an unlabelled host
You can track where the GFP expressing cells end up later in development
What is a benefit of fat napping with transgenic DNA?
Dilution is not an issue
Describe transgenic memory cassettes lineage tracing
Genetically engineered constructs that undergo permanent, heritable modifications in response to specific biological stimuli, allowing tracing of self fate overtime
Describe in situ hybridization
Allows you to visualize RNA expression in the whole embryo
What type of embryos is in situ hybridization performed on?
Fixed embryos
What does in situ hybridization tell you?
Tells you where RNA is expressed but not how much is being expressed.
Describe immunohistochemistry
Allows for visualization of protein expression pattern in the whole embryo
What information does immunohistochemistry provide you with?
Tells you where protein is expressed, but not the amount
Describe loss of function genetics
Eliminate gene function partially or completely
What does does loss of function genetics show?
Necessity
Describe gain of function genetics
Creates conditions where a gene is excessively or ectopically expressed or its function is exaggerated
What does gain of function genetics show?
Sufficiency
Describe forward genetics
Phenotype to genotype
Looks to identify the genes responsible for a particular biological process or function in an organism
Describe reverse genetics
Genotype to pheno type
Looks to unravel functions behind genes of interest
What does RNA sequencing do?
Sequences and quantifies DNA in a cell
Outline the four steps of RNA sequencing
A list of genes expressed in cells of a specific stage, tissue or mutant background is provided
You can compare lists from your tissues/cells of interest and related tissues/cells
Identify genes that are differentially expressed in cells of interest and further narrow down the list with bioinformatics
And then analyze your candidates with loss of function/gain of function experiments
What part of the genome is of interest in creating transgenic organisms?
Enhancers
What do enhancers do?
Control wear genes are expressed
What is by enhancers being modular?
They can act on their own if they are put up stream of another gene
Outline how a transgenic organism is created
Scientists can make recombinant DNA constructs in the lab in which an enhancer is cloned upstream of another gene
The constructs are made with PCR and restriction enzymes
The constructs are then integrated into an animal
Describe CRISPR/Cas9-mediated gene editing?
Allows researchers to mutate any gene or insert reporters into endogenous loci
How does CRISPR work?
A stretch of DNA that went transcribed produces guide RNA that recognize segments of viral DNA
Describe the process of CRISPR/CAS9-mediated gene editing
Guide RNAs and CAS9 inserted into genome
CAS9 causes double strength breaks
A portion of DNA is cut out
This leads to a premature stop codon and loss of function
