1. C. Elegans

2. Purpose of the Lab To learn about DNA
Inject DNA into living organisms in an attempt to have the offspring express the traits
Stop gene silencing
OVERALL: Develop a mechanism to put genes into the germline of the organism so they are passed down (successful transformation)

3. Background Dr. Mello DNA Homologous Recombination Flanking Sequences
Extra-Chromosomal Arrays
DNA Silencing
Plasmids
C. Elegans
Body Structure
Germline/genome

4. Dr. Craig Mello Nobel Prize for RNAi
Attempting to reinsert genes into their locus, and have them expressed in later generations

5. DNA 4 bases Double stranded Genetic material Chromosomes
Genes
Chromosomes
DNA replication
Genome

6. Homologous Recombination
Meiosis
DNA fixes itself
Double Stranded break
Takes the DNA from the sister chromosome to fix itself
Ends up with recombined DNA
Gene targeting

7. Flanking Sequence Short sequences that surround the gene of interest
Usually do not code for anything
Used in homologous recombination to determine the area to be copied
Match on each chromosome
Used to insert gene of interest

8. Extra-Chromosomal Arrays
DNA which exists outside the common chromosomes
Usually not integrated into DNA
Prone to gene silencing
Not stable
Injected plasmid is copied at a high number, need low copy number to pass on to offspring

9. DNA Silencing RNAi silences Used to protect DNA from viral infections
Protect DNA for outside influences
Usually stops multi-copy
Stops extra-chromosomal arrays from incorporating into DNA permanently

10. Plasmids Used to inject wanted gene into the organism
Contains gene of interest, flanking sequences, selectable marker, counter-selectable marker
Used in homologous recombination

11. C. Elegans Nematode worm Simple body structure Reproduce quickly
Similar chromosomes to humans
DNA easily injected into adult worm’s germline
Can be mass produced

12. Body Structure

13. Genome/germ-line Inject DNA into gonads
Where the sperm/ovaries are located
Where the DNA will come from for children
2 arms in C. Elegans, with a turn

14. Procedure
Create the plasmid containing the gene of interest and the transposase which will cause the DNA to break
Inject into about 50 worms to ensure some success
Let the worms reproduce, checking each generation
If successful, the later generations should express the gene of interest

15. DNA Injection
Movie

16. Transposons
Movie

17. Transposase Moves transposons from one area on the genome to another
Can be cancerous
Binds to the end of transposons and facilitates their “jumps”
Injected with the plasmid
Causes double stranded breaks
Allows the gene of interest to be taken from the plasmid

18. Plasmid fixes DNA
Double stranded break due to the injected transposase
DNA seeks to repair itself
Plasmid has the same flanking sequence as the gene that “jumped”
Homologous Recombination

19. Glh-2 Used to express the Mos transposase
Expressed naturally in C. Elegans at all stages of life
Germ-line specific
Along with Glh-1 required for normal germline development
Recognized by the cell so not silenced

20. MosSci (Mos Mediated Single Copy Insertion)
In C. Elegans, Mos genes have been inserted throughout the DNA, but they express no characteristics
Inject Mos transposase to make it “jump”
Know the flanking sequence, so able to match gene of interest to locus
Less chance of silencing (No extra-chromosomal arrays)
Expressed under glh-2 promoter
Used in unc-119 rescue (no RFP)

21. Unc-119 Needed for proper development of the nervous system
Paralyzed worms (marker)
Neuronal gene (less likely to be silenced than a germline gene)
Start with unc strain and then rescue with plasmid, those that move contain gene of interest

22. RFP/GFP Found in jellyfish Seen through UV microscope
Injected into worm to mark it
Those that express also express gene of interest
Same plasmid and in same sequence

23. Rollers http://130.15.90.245/movies/C.%20elegans%20Roller%20Mutant.mov
Injected with DNA with makes their bodies uncoordinated
Roll around their axis
Helically twisted body
Used as a marker, those that express have gene of interest

24. Heat Shock 34ºC Enhances expression down stream
Instead of glh-2 (takes a week longer)
Helps the proteins fold at a higher temp
Plasmids assemble
Inject 10, grow 1000 offspring
Too much heat, worms paralyzed (Twk)

25. Counter-Selectable Vector
Outside the gene
Example: avr15
Worms injected with plasmid that codes to be Ivermectin prone
Worms were previously immune to ivermectin
Placed on plate, those that die have incorporated DNA that was not wanted, but the majority should not uptake any as it is now with the gene of interest

26. Ivermectin Used to kill nematodes
Used to test counter-selectable vectors
Used as gene of interest to test the ability to knock out proteins
If the worm lacks three genes, avr15, avr14 and glc-1, then it is immune
Perfect for lab environment
Not perfect in wild
elegans.swmed.edu/ECWM/2002/abstracts.pdf pg 263

27. Transformation Uptake of foreign DNA
Leads to the change in genetic information passed down to offspring
Difficult for the genes not to be silenced
Does not usually succeed
Change in genetic information expressed

28. Ribosomal Gene Drosphilia family 50 nucleotides long
Used as a selectable marker (inside the gene, don’t need expression)
Small enough not to interfere with the gene

29. MicroRNA No marker is needed for the insertion of the gene of interest
Needs to be a very small selectable marker
Tiny RNA --> functions via RNAi pathway
21 nucleotides (gene = nucleotides)
Used so it doesn’t interfere with gene expression

30. Restriction Enzyme ISCF1 has a long recognition sequence which is rare
Cut flank region of interest
Cuts double stranded DNA
Defense against viruses
Used for DNA modification

31. Zinc Finger Nuclease Lab generated restriction enzymes
Zinc finger DNA-binding domain which is fused to the cleavage domain of the FokI restriction endonuclease
Target specific DNA sequences
Recognize any sequence
Specialize to target any part of the gnome
Downfall: need to engineer different nuclease for each gene