Molecular Tools in Developmental Biology

Question 1

We will be studying techniques that allow us to do two things:

Answer 1

1) Assess when and where a gene is expressed

2) Perturb gene expression or function

Question 2

What is the easiest way to study a protein?

Answer 2

Work with the DNA that encodes it.

Question 3

What is the difference between [DNA and tRNA] vs. [mRNA]?

Answer 3

mRNA does not have any introns.

Question 4

How do you make cDNA?

Answer 4

You use reverse transcriptase to create a copy of mRNA. This is useful in studying a protein.

Question 5

What is Nucleic Acid Hybridization useful for?

Answer 5

You can create strands that are complement to the ones that you want, in order to probe for them.

Question 6

Describe in Situ Hybridization

Answer 6

You create a cDNA with a dioxygenin on it. This cDNA is complement to the mRNA you want to find. Finally, you can use an antibody as a probe that looks for the dioxygenin, and hence the mRNA.

Question 7

Describe Indirect Immunostaining

Answer 7

You add primary antibodies that connect to ANY molecule of interest. You then can add fluorescent secondary antibodies to identify the molecule of interest.

Question 8

Describe tagging a protein with GFP

Answer 8

You can add a piece of DNA that creates GFP so that when a molecule is created, it has a small fluorescent tail piece.

Question 9

What is confocal microscopy useful for?

Answer 9

Eliminating out of focus fluorescence from thick specimens like embryos.

Question 10

Over expression of mRNA tests for?

Answer 10

sufficiency

Question 11

Removing the function of a gene or protein tests for?

Answer 11

Necessity

Question 12

What two removal methods acts at the level of the mRNA?

Answer 12

anti sense oligonucleotides, and RNAi

Question 13

What removal method acts at the level of proteins?

Answer 13

morpholino antisense nucleotides

Question 14

How does RNAi work?

Answer 14

Short strands of RNA create RISC complex that search and destroys normal mRNA. These short strands can either directly be siRNA’s, or can be formed by introducing double stranded RNA.

Question 15

How do morpholinos work?

Answer 15

These chemically modified oligonucleotides bind to and prevent translation of mRNA

Question 16

What are the four types of modification at the DNA level?

Answer 16

Transgenics, homologous recombination, CRISPR/Cas9, and mutations.

Question 17

What is it called when you add genetic material?

Answer 17

Transgenics

Question 18

Describe the process of homologous recombination?

Answer 18

Create piece of DNA and insert at gene site via recombination.

Use double drug selection to identify ES cells with said DNA exchange.

Place these cells into chimeric embryo

Breed until you get homozygous knockouts.

Question 19

What is CRISPR/Cas9 good for?

Answer 19

Replacing specific DNA

Question 20

What is the “Maternal Effect” when it comes to mutants?

Answer 20

When the mother is mutant, the offspring have a phenotype regardless of their own genotype. This is because the mother donates mRNA and proteins to the egg.