C. Elegans

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)

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

Dr. Craig Mello Nobel Prize for RNAi Attempting to reinsert genes into their locus, and have them expressed in later generations http://cache.daylife.com/imageserve/0aeI9WE6gkauc/340x.jpg

DNA 4 bases Double stranded Genetic material Chromosomes Genes Chromosomes DNA replication Genome http://ghr.nlm.nih.gov/handbook/basics/dna

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 http://www.bioscience.org/1998/v3/d/bianco/fig1.jpg

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

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

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

Plasmids Used to inject wanted gene into the organism Contains gene of interest, flanking sequences, selectable marker, counter-selectable marker Used in homologous recombination http://www.sciencev3d.org/bilder/plasmid.jpg

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 http://www.wormclassroom.org/ce/Kahn/C.elegans_L4.jpg http://blogs.zdnet.com/emergingtech/?p=211

Body Structure http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Caen.elegans.html http://www.loci.wisc.edu/outreach/text/celegans.html

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 http://www.wormbook.org/chapters/www_transformationmicroinjection/WMTransformfig1.jpg

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

DNA Injection Movie

Transposons Movie

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

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 http://www.biology.duke.edu/model-system/ymsg/cloning.html

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 http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=worm&q=glh-2

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) http://sites.google.com/site/jorgensenmossci/protocols

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 http://www.wormbase.org/db/gene/gene?name=WBGene00006843;class=Gene

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

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 http://www.citeulike.org/user/tharris/article/3188077 http://www.genetics.org/cgi/content/abstract/95/2/317

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)

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

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

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

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 http://www.biobasics.gc.ca/english/View.asp?x=696&mid=427

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

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

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 http://www.zincfingers.org/scientific-background.htm