VL6 - C.elegans II

Question 1

RNA interference (RNAi)

In c.elegans

Answer 1

Craig Mello and Andrew Fire - Nobel prize 2006

• refers to the knockdown of gene expression by small RNA fragments
• controlled by RNA-induced silencing complex (RISC) and is initiated by
  short double-stranded RNA (dsRNA) molecules in a cell’s cytoplasm, where they interact with the catalytic RISC component Argonaute.

Process
1. Cleavage of dsRNA in shorter dsRNA-DICER
2. Loading short single stranded RNA into a ribonucleoprotein complex - SLICER
3. Scanning for target RNAs in the cell

https://www.youtube.com/watch?v=cL-IZnpY6Qg

Question 2

Why is c.elegans an ideal system for studying gene expression and function?

Answer 2

• developmental timeline of every cell has been mapped
• Microinjection technique allows direct introduction of macromolecules into animals (RNA, DNA, Proteins)
• ability to affaect large population

Question 3

Antisense vs RNAi

Answer 3

Antisense Technology:
* Principle: In antisense technology, synthetic nucleic acid sequences (antisense oligonucleotides) are designed to be complementary to specific messenger RNA (mRNA) sequences.
* Mechanism: When introduced into cells, antisense oligonucleotides bind to the complementary mRNA, forming a double-stranded RNA structure.
* Effect: This binding inhibits the translation of mRNA into proteins, leading to reduced expression of the targeted gene

RNA interference (RNAi):
* Principle: RNAi is a natural cellular process that regulates gene expression by using small RNA molecules, such as small interfering RNA (siRNA) or microRNA (miRNA).
* Mechanism: Small RNA molecules bind to target mRNA, leading to the degradation of the mRNA or inhibition of its translation.
* Effect: RNAi can specifically silence or “knock down” the expression of genes, providing a powerful tool for studying gene function and potential therapeutic applications

Discovery:
* Researchers initially explored the idea of using synthetic antisense oligonucleotides to specifically bind to and inhibit the expression of target messenger RNA (mRNA).
* The concept involved designing complementary nucleic acid sequences to selectively interfere with the translation of specific mRNAs into proteins.
* The breakthrough in RNAi came with the work of Andrew Fire and Craig Mello in 1998. They were investigating gene silencing in the nematode Caenorhabditis elegans.
* Fire and Mello introduced double-stranded RNA (dsRNA) into C. elegans, expecting it to act as an antisense inhibitor.
* However, they observed a more potent and systemic gene-silencing effect, which was later termed RNA interference.

Question 4

exogenous RNAi vs. endogenous RNAi

Answer 4

exogenous RNAi
* infection by a virus with an RNA genome
* laboratory manipulations

endogenous RNAi
* micro RNAs (miRNAs)
* small interference RNAi (endo-siRNAs)
* Piwi-interaction RNAi (piRNA)

Question 5

Why is RNAi important?

in c.elegans

Answer 5

• provides valuable insights into eveolution
• found in all eukaryotes (except some fungi)
• indicates that gene expression is the key to evolving complex organisms

BUT: Invaluable tool for functional genomics
–> Knowckdowns are easier than knockouts, saves time and money

Knockdown:
a reduction in the expression or activity of a gene, typically achieved by suppressing the production of its mRNA or inhibiting translation. (RNAi)

Knockout:
complete elimination or inactivation of a gene, typically by disrupting its coding sequence or regulatory elements. (CRISPR-Cas9)

Question 6

Transgenic lines

Answer 6

• transgenic lines are created through genetic engineering techniques, where foreign genes (transgenes) are inserted into the genome of the organism.
• any organism whose genetic material has been altered using geetic engineering (S1 or higher)

Structure
1. Promotor
(endogenous, tissue specific, inducible promoter e.g Heat shock)
2. Target gene
–> specific gene of interest that researchers aim to study, modify, or express in the transgenic organism. PCR Fragment or Plasmid
(endogenous, sensors eg: pH sensetive)
3. Tag (or marker gene)
introduced into an organism alongside the target gene to serve as a visible or selectable marker. Helps identify or select cells or organisms that have successfully incorporated and expressed the introduced genetic material. Marker-Plasmid
(small TAgs, fluorescent tags, no tag)
4. 3´UTR

Question 7

GFP

Answer 7

Green fluorecent Protein
Osamu Shimomura, Martin Chalfie and Roger Tsien - Nobel prize in 2008

1. Osamu Shimomura:
discovered GFP in jelly fish and its crystal structure
2. Douglas Prasher (forgotten in Nobel prize)
Cloned and sequenced GFP –> shared info with Tsien and Chalfie
3. Martin Chalfie:
Used GFP as a marker for gene expression in E.coli and c.elegans
4. Roger Tsien
Showed that GFP is formed in chemical reaction which needs oxygene
Made various GFP mutants and created monomeric variants which can glow pink, red, orange

Question 8

Whole genome sequencing in C.elegans

Answer 8

• 1998 John Sulstona nd Alan Coulson finished their whole genome project
• 20444 protein coding genes mapped
• Most genes relativly small (compared to vertebrate genes) ca. 3kb
• Introns make up to 26% of the genome
• Intergenic regions make up 47% of the genome

Question 9

C.elegans in Agning

Answer 9

• good because of short life span (18-20 days after 3 days of development)
• Agning characteristics include (Lipofuscin aggregates, Muscle degenartion, Neuronals deficiency, Vacuole like structrue, oxidized proteins)
• great variation in agning

--> stress resistance (UV radiation, heat, hypoxia, heavy metals) = longevity

C.elegans serve as an excellent model organism for cell fate and developmental mechanisms