Genetically accurate models of SOD1-based ALS in Drosophila

NDALPınar Deniz23/11/2010

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OUTLINE

Amyotrophic Lateral Sclerosis (ALS)

• Neurodegenerative & complex disorder

– death of motor neurons in the brain, brainstem, and spinal cord, resulting in fatal paralysis.

• The majority of ALS is acquired spontaneously, (Sporadic ALS)

• Inherited type of the disease (Familial ALS ) accounts for only 10% of all cases (fALS).

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20% of familial cases!

ALS

Genetic causes of ALS

• Neutralize superoxide radicals, which can damage cells if their levels are not controlled.

• To function properly, the superoxide dismutase enzyme must bind to Cu & Zn.

The human SOD1 enzyme

•32-kDa homodimer

•153 amino acid

• 5 exons

SOD1

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Pasinelli P., Brown R. H. 2006

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SOD1 conservation

NCBI, HomoloGeneINTRODUCTIONMETHODS FUTURE GOALS

Modelling SOD1 mutants

• Small• Cheap• Short generation time

As a model for neurodegenerative diseases;

complex nervous system-displays complex behaviours: learning and memory

particularly attractive system for studying neurodegenerative diseases.

Fruit Fly?

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Over-expression? Knock-out?

• Knock-in by homologous recombination (HR)

– preserves endogenous gene regulation and ensures that pathological changes in cell function and physiology result only from the effects of the desired mutation

More suitable for studying complex neurodegenerative disorders such as ALS.

Knock-in?

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Human SOD1

Drosophila SOD

G37R H48R H71Y G85R

9309 KB

1457 KB

Knocking-in: SOD mutations

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Introducing dSOD mutations by HR

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Homolog arms

- generated from fly gDNA by PCR

- start/end in areas outside of the coding sequence

-~2.5 kb

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Restriction sites are added by primersduring PCR

PCR products TOPO cloning

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Homolog arms

Digestion verification

Sequencing

Preparing the mutantarms

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Homolog arms

Dropping out the arms fromTOPO with appropriate REs

Cutting pw25 with the samerestriction enzymes

Ligating the first arm in pw25

Transformation

Mini prep

Digestion of mini prep DNAs with the same REs

Junction sequencing (FRT-SceI-Lox P sites)

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Insert construct in fly genome

Cre-lox P recombination

Give the fly “red” eye colorLinearize theexcised part

FLP-FRT recombination:Excise the construct

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INTRODUCTIONMETHODS FUTURE GOALS

Microinjection!

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w 1118 males

No excision Red eye

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Heat shock

Flp effect

Excision white / mosaic eye

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Linearized by I-Sce

Transgene is mobilized by ey-FLP

Not collected

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eyFLP: To distinguish betweenred eyed flies

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w 1118 males

Sacrifice the fly gDNA

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• Suppress recombination • Carry dominant markers • Lethal, when carried homozygously.

Cre flies: Remove the mini white gene

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White eye due to the loss of mini w gene.

Post Cre validation

Product from targeted chromosomewill cut with AscI and Acc65I

Product from the endogenouschromosome will not cut with AscI andAcc65I

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What is next?

Assessing the phenotypic consequences.

-Taking the advantage of short life span of fruit flies, thecorrelation of chronological age with symptom severity will berapid compared to mouse studies.

Observed phenotypes will be used to inform downstreamcharacterization.

- For instance, there are a number of simple and rapid stress testswhich can and will be performed on the mutants generated inthese studies.

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Acknowledgments• Nazlı Başak & NDAL team

• Robert Reenan & Reenan lab members

• Arzu Celik’s lab Ece Terzioğlu Kara &Sercan Sayın

• All MBG members

THANK YOU