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Construction of an Usher Syndrome Type IIIb Blindness and De

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Shannon Lozito

on 6 May 2014

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Transcript of Construction of an Usher Syndrome Type IIIb Blindness and De

Zebrafish Procedure Overview
Embryos are injected immediately after being laid.
Enbryos are de-corionated after a specific period of development (28 hours or 5 days in these particular experiments).
The yolk sac is removed (using a pipette for 28 hour fish and manually for 5 day fish).
Preparation of mRNA
Designed and diluted primers HARSdr and HARSrev according to directions on the oligonucleotide data sheet.
Checked dilutions to find the concentration of nucleotides using Nanodrop technology.
Used primers to amplify the gene using the plasmid.
Checked PCR by sequencing. The bases changed were confirmed using data in other complimentary strands. Both synonymous and non-synonymous mutations in sequence were confirmed.
Used Ampliscribe kit to transcribe mRRNA
Current Situation
The arrows represent that the same signal is observed for the uninjected control and the HARS and Y454S mutant flag. This could be for one of three reasons:
1. Primers did not amplify the sequence properly
-NO because our protein product has the complete FLAG tag
2. mRNA degraded
NO-checked using PCR
3. Insufficient amount of mRNA was injected
-injecting more mRNA in the current experiment
Future Projections
Examining organ structure and cell morphology in the eye, ear and neuromast (sensory cells of the lateral line).
Immunofluorescence and confocal microscopy will be used to identify co-localization of HARS with proteins identified by the proteomics screens, and cellular localization of the HARS mutant protein.
Specific marker antibodies such as cadherin, which is found in hair cells of both the ear and eye, and neurofilament, to mark neural cells, will be used in imaging.
Observe behavior: the fish require functional lateral line neuromasts and hair cells in the semicircular canals for directional swimming.

Construction of an Usher Syndrome Type IIIb Blindness and Deafness Model in Zebrafish

Shannon Lozito

With Yolk sac
Yolk sac removed
1. To prepare the mRNA and inject
the fertilized zebrafish eggs with human HARS or mutant HARS mRNA.
2. To verify the gene expression of
the protein to confirm the success of the injections using quantitative PCR.
3. To analyze where the HARS protein
appears in zebrafish mechanosensory cells for both control and mutant fish.
4. To assess the behavior of the
zebrafish due to the effects of HARS on the sensory cells.

PCR Product
HARS and y454S mRNA PCR
100 bp
1 kb
0.5% agarose gel
1.8% agarose gel
putative vector
putative PCR product
Western Blots
Amplification of gene from plasmid was successful-received DNA
In-vitro transcription was successful
Decided to remove yolk sac due to streaking
Expression of HARS can be seen in as little as 0.25 fish/well.
Due to a difference in molecular weight, fish and human HARS can be differentiated.
Gel 1: 28 hour
L: mutant R: wt
Gel 2: 28 hour UIC
Gel 4: 5-day UIC
Gel 3: 5-day
L: wt R: mutant
Full transcript