7. VL

Question 1

RNA interference?

Answer 1

Process silence gene, refers to knockdown gene expression by RNA fragments, phenotypes are easy to spot (movement), post transcriptional gene silencing,
occorse via siRNA, first seen in C. elegans

Question 2

step by step RNAi?

Answer 2

dsRNA trigger at a RNA

1. : RDE-4 binds dsRNA &recruits RDE-1
2. : RDE-4 & RDE-1 recruit DCR-1 (Dicer)
3. :Dicer degrades dsRNA to short- interfering RNA (siRNA)
4. : siRNA guide strand incorporated into RNA Induced Silencing Complex (RISC) - RISC mit antisence, slicer is part of RISC component
5. : RISC binds target mRNA via base pairing
6. : Slicer degrades mRNA
7. :cut between nucleotides 10 & 11 from 5’ end

Question 3

RNAi - why important?

Answer 3

play a role in evolution (Found in all eukaryotes except some fungi -> ancient biochemical mechanism), invaluable tool for functional genetics (Knockdowns are easier than knockouts)

Question 4

C. elegans - why ideal for gene expression?

Answer 4

developmental timeline of every cell is mapped, phenotypic response to gene expression in well defined, microinjections: direct injection of macromolecules, ability to affect large populations, RNA/ DNA and proteins can all be injected

Question 5

How had been found out, which strand of RNA is responsible for the twitching movement of C.elegans’ offspring? (!!!!!!!)

Answer 5

double strang RNA can come in the worm by injection or bring them into E.coli (which are the food of C. elegans)

Question 6

in which other organisms dsRNA triggered silencing?

Answer 6

Drosophila, plant systems, not mammalia

Question 7

success of experiment of Andrew Fire and Craig Fire?

Answer 7

pos1 RNAi (knockdown) -> animals die, mutagenesis of the RNAi -> C. elegans survived. Genes of mutagenesis: rde-4 and rde-1 (part of RNA interference)

Question 8

What is special about RNAi in C. elegans? why? (!)

Answer 8

an extra step: , increases the efficiency

Question 9

What is osmoregulation? what gene is involved?

Answer 9

regulation the salt concentration and water (pressure), osm-8, osm-8 mutation –> gpdh-1 upregulation (!) but a ptr-3 RNAi can rescue the phenotype

Question 10

What is Transgenic animals - genetically modified organisms (GMO)?

Answer 10

Any organism whose genetic material has been altered using genetic engineering techniques.
•In Germany, GMOs are classified as S1 or higher, depending on the DNA inserted.

Question 11

How do you design the gene transgenesis DNA?

Answer 11

1. promotor (endogenous, tissue specific, inducible promotor, e.g. heat shock) 2. ATG and target gene (endogenous, sensors, e.g. pH sensitive, Ca2+ sensitive) 3. tag (small tags, fluorescent tags (GFP, RFP) no tag)

Question 12

How does transgenesis work?

Answer 12

2 preparing steps: 1: creation of the vector; 2. preparing eucaryotic chromosome
Vector: cloning or expression if a vector, digestion with restriction endonuclease
Eucaryotic chromosme: PCR of OR target gene of interest, digestion with restriction endonuclease, DNA fragment contain gene of interest
LIGATION: recombinant plasmid

Question 13

where does microinjection takes place in C. elegans?

Answer 13

distal over the gonades

Question 14

Some facts about GFP - why do we talk about GFP during C.elegans lecture?

Answer 14

238 Amino acid residues; approx. 27 kDa
• Fluorophore of GFP is build autocatalytically from the tripeptide sequence Ser65–Tyr66–
Gly67 within the polypeptide chain.
• absorption maxima: 395 nm / 475 nm
• emissions maximum: 509 nm
GFP has first been used for gen expression marker in C. elegans (1994)
other colors (DsRED, orange…)

Question 15

What are extrachromosomal arrays?

Answer 15

if transgenesis does not work: non-integrated transgenic line, the transgene will be present as an extrachromosomal array - a mini chromosome.
• Non-integrated transgenic lines need a lot of maintenance and can get lost or silent after
several generations.
Integration of extrachromosomal arrays is possible via:
UV integration, gamma irradiation (but hard to do)

Question 16

Solution for extrachromosomal arrays? (step by step ab Folie 36)

Answer 16

Single copy integration
Method developed by Christian Frokjaer-Jensen and colleagues (2008), Allows integration of a single copy at a defined location, Variation of mutagenesis via MosI transposon.

Question 17

what is physical mapping?

Answer 17

molecular biology techniques to examine (untersuchen) DNA molecules directly in order to construct maps showing the positions of sequence features, including genes. 2 methods: Cosmid library (smaller pieces), YAC (yeast artificial chromosomes, only infect yeast cells, no bacteria) to fill the gaps between pieces (small and long pieces) - how methods work: in the slides no 43 -

Question 18

since when whole genom sequencing id possible?

Answer 18

whole genome sequencing in 1992, Sulston and Waterston were able to sequence 1.000.000 base pairs per year, 1995 they were able to sequence several millions of base pairs per year.

Question 19

How does antisense mediated silencing (popular in 1990’s) worked?

Answer 19

target mRNA (should be silenced) , many copies of mRNA complementary RNA, translation inhibition (mRNA degradation) , target gene silenced