Quiz #2 (Module 2), First 1/2 (Experimental + Techniques)

Animal Models

Used in biomedical research because it can mimic aspects of a biological process or disease found in humans. Sufficiently”like-humans” in their anatomy, physiology, or response to pathogen so that the results can be extrapolated to better understand human physiology and disease.

Factors to choosing a good model

1) Can you keep them and breed them in a lab at a reduced cost?

2) Amendable for genetic manipulations?

3) Short generation time?

4) High fecundity? (produce abundance of offspring)

C.elegans

Very cheap, breed easily (RNAi, knock down genes and note resulting loss-of-function phenotype), 3-5 days for sexual maturity, lay 4-10 eggs/hour

Macaca mulatta

Sexual maturity (3-4 years), possible to do transgenesis/CRISPR-Cas, ~ 1 baby in each pregnancy (gestation 5-6 months)

What is the relationship between Throughput and Biological Complexity/Cost in research models?

Scale of experiments (thousands of samples vs a few) vs similarity to us

Funnel goes from top to bottom with organisms in what order?

Cells (in-vitro) → organoids/3D cultures → C.elegans → Drosophila → Zebrafish → Mammals (Mice & Monkeys)

Zebra fish share about _% of the same genes as humans.

70

The protective layer that encloses the yolk of a zebrafish egg?

Chorion

Find it experiment looks for

Where (localize) and when a gene is expressed. Examples) In Situ Hybridization

Lose it experiments look for

Removing/blocking a gene and seeing if that causes a loss of function. Examples) CRISPR and RNAi

Move it experiments look for

See if a gene itself is enough to trigger the process in a completely different location. “Gain of function”.

alcian blue stains for

cartilage

alizarin red stains for

bone

Whole mount staining → in toto

entire thing, take animal → freeze, introduce different dyes

H & E histology

Hematoxylin & Eosin, wont give an identity to the cells, wont say what is cartilage and what is bone

Hematoxylin

Stains mostly the nuclei blue/purple since DNA is an acid and a toxin is a base

Eosin

Stains cytoplasm/extracellular matrix pink/red

Trichrome

Basically same as Pentachrome

Pentachrome

Five different dyes that will result in 5 different colors, which allows to see different structures of different complexity.

The Central Dogma

Describes how genetic information flows in a cell

The Central Dogma Process

Transcription (DNA is copied into pre-mRNA in the nucleus) → Processing (pre-mRNA is modified into mature mRNA) → transport (mRNA moves out of the nucleus into the cytoplasm) → translation (a ribosome reads the mRNA to create an amino acid chain) → protein folding and modification (the chain is folded into a functional 3D shape) → carry out function (the finished protein performs its specific biological role)

At what point of the central dogma process would we look at the mRNA?

during the processing step (now mature mRNA)

To detect mRNA

in-situ hybridization, this technique uses an anti-sense probe (critical reagent) to bind to critical regions of the mRNA

To detect Proteins

Use Immuno-fluorescence, this technique uses an antibody that specifically recognizes and binds to the target protein

DAPI

Specifically stains the cell nuclei by binding to DNA, DAPI is not an antibody, chemical stain that “gets into the groove” of DNA. Simply a reagent that will bind to nuclei, help looks at the landscape.

How does an antibody show color in Immunofluorescence?

A specific antibody recognizes and binds to its target protein (e.g, SOX2 or ISL1) → antibody is labeled with a fluorophore —> once hit with light, the fluorophore “gets excited” and emits color

Do antibodies themselves have color?

No, which is why they are labeled with a fluorophore, which once hit with light will get excited and have higher energy and return to a basal state (return is that release of light)

Most of the time In Situ hybridization is used over Immunofluorescence because …

a specific antibody has not been developed or is not present for a target protein

In situ hybridization

A technique used to visualize the location of specific genetic information before it becomes a protein, using a complementary (anti-sense) probe that binds to the target mRNA sequence. Showing exactly where a gene is “turned on” in a tissue. His definition: “A technique to to detect the transcription of the gene, the mRNA”.

Histology

Basically fixing the animal → put in some sort of media (sometimes wax, called paraffin) or a block of something you can freeze → then you will section → then stain

mRNA is extremely sensitive to

degradation

in RNA, Thymine is replaced with

Uracil (U)

The anti-complementary probe will go

5’ to 3’

Key reagent in, in-situ hybridization is the

antisense complementary probe

If you look the mRNA it will go from 5’ to 3’ (left to right) , so anything that is complementary will be reverse

going 5’ to 3’ (right to left)

the specificity of the strand and complementary strand of mRNA is produced by the

sequence

We can produce any probe that has a specificity to a gene based

on the sequence

anti-dig in the example is attached to a uracil and is conjugated (attached) to an

enzyme called alkaline phosphatase (AP) which will catalyze a reaction in the presence of a substrate that doesn’t have any color, that will then produce a product that is purple. Not flourescent, but color produced will stay in there.

The anti-sense probe is recognizing the

gene of interest, that will then will result in a reaction (the alkaline phosphatase) that will result in purple.

Image appears to be in purple?

Must be an in-situ hybridization

Most of the mRNA of a cell will be in the

cytoplasm (mature form of the RNA (mRNA) will be exported into the cytoplasm)

Many genes are expressed in a _ and very _ manner

cell-specific; dynamic