C. Elegans. Purpose of the Lab To learn about DNA Inject DNA into living organisms in an attempt to...
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Transcript of C. Elegans. Purpose of the Lab To learn about DNA Inject DNA into living organisms in an attempt to...
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
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DNA
4 bases
Double stranded
Genetic material Genes
Chromosomes
DNA replication
Genome
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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
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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
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C. ElegansNematode wormSimple body structureReproduce quicklySimilar chromosomes
to humansDNA easily injected
into adult worm’s germline
Can be mass produced
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Body Structure
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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
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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
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
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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
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)
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
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
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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
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
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
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