DevBio Toolkit

Question 1

List the seven parts of the tool kit discussed in class

Answer 1

Lineage tracing, in situ, hybridization, immunohistochemistry, genetics, deep sequencing (RNA), transgenic organisms, CRISPR/Cas9-mediated gene editing

Question 2

List the four different types of lineage tracing.

Answer 2

Direct observation, fate mapping using dyes, fate mapping with transgenic DNA, transgenic memory cassettes.

Question 3

What is required for direct observation lineage tracing?

Answer 3

Organisms with unique cells that retain their uniqueness throughout development.

Question 4

What are the two different types of fate mapping using dyes?

Answer 4

Vital staining and fluorescent dye.

Question 5

Describe vital dye staining

Answer 5

Introduce dye into a group of cells early, see which cells are stained later in development

Question 6

What is required for vital dye staining lineage tracing?

Answer 6

Being able to open the embryo or having a somewhat transparent embryo

Question 7

What is a drawback of vital dye staining?

Answer 7

The dye dilute as cells divide

Question 8

How is fluorescent dye different than vital dye staining?

Answer 8

Fluorescent die does dilute, but it is visible regardless of embryo transparency

Question 9

Describe using genetic markers as cell lineage tracers

Answer 9

Transplant a cell from a similar organism to the organism of interest in its equivalent location

Question 10

How is lineage tracing visualized through genetic markers?

Answer 10

Based on morphology or using antibodies

Question 11

Why are genetic markers as so lineage tracer no longer common practice?

Answer 11

You need similar organisms, time intensive, not used in utero

Question 12

Describe fate mapping with transgenic DNA.

Answer 12

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

Question 13

What is a benefit of fat napping with transgenic DNA?

Answer 13

Dilution is not an issue

Question 14

Describe transgenic memory cassettes lineage tracing

Answer 14

Genetically engineered constructs that undergo permanent, heritable modifications in response to specific biological stimuli, allowing tracing of self fate overtime

Question 15

Describe in situ hybridization

Answer 15

Allows you to visualize RNA expression in the whole embryo

Question 16

What type of embryos is in situ hybridization performed on?

Answer 16

Fixed embryos

Question 17

What does in situ hybridization tell you?

Answer 17

Tells you where RNA is expressed but not how much is being expressed.

Question 18

Describe immunohistochemistry

Answer 18

Allows for visualization of protein expression pattern in the whole embryo

Question 19

What information does immunohistochemistry provide you with?

Answer 19

Tells you where protein is expressed, but not the amount

Question 20

Describe loss of function genetics

Answer 20

Eliminate gene function partially or completely

Question 21

What does does loss of function genetics show?

Answer 21

Necessity

Question 22

Describe gain of function genetics

Answer 22

Creates conditions where a gene is excessively or ectopically expressed or its function is exaggerated

Question 23

What does gain of function genetics show?

Answer 23

Sufficiency

Question 24

Describe forward genetics

Answer 24

Phenotype to genotype
Looks to identify the genes responsible for a particular biological process or function in an organism

Question 25

Describe reverse genetics

Answer 25

Genotype to pheno type Looks to unravel functions behind genes of interest

Question 26

What does RNA sequencing do?

Answer 26

Sequences and quantifies DNA in a cell

Question 27

Outline the four steps of RNA sequencing

Answer 27

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

Question 28

What part of the genome is of interest in creating transgenic organisms?

Answer 28

Enhancers

Question 29

What do enhancers do?

Answer 29

Control wear genes are expressed

Question 30

What is by enhancers being modular?

Answer 30

They can act on their own if they are put up stream of another gene

Question 31

Outline how a transgenic organism is created

Answer 31

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

Question 32

Describe CRISPR/Cas9-mediated gene editing?

Answer 32

Allows researchers to mutate any gene or insert reporters into endogenous loci

Question 33

How does CRISPR work?

Answer 33

A stretch of DNA that went transcribed produces guide RNA that recognize segments of viral DNA

Question 34

Describe the process of CRISPR/CAS9-mediated gene editing

Answer 34

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