C. Elegans

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C. Elegans

description

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). - PowerPoint PPT Presentation

Transcript of C. Elegans

Page 1: C. Elegans

C. Elegans

Page 2: 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)

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Background

Dr. Mello DNA Homologous Recombination Flanking Sequences Extra-Chromosomal Arrays DNA Silencing Plasmids C. Elegans

Body Structure Germline/genome

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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

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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

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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

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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

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DNA Injection

Movie

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Transposons

Movie

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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

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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

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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)

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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