C. Elegans

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 • 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

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 = 300-400 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