Academic Field

Biological Sciences

Faculty Mentor Name

Adam Rich

Presentation Type

Oral Presentation

Abstract

Morpholino oligonucleotides (MO) are commonly used to reduce gene expression. MO selectivity is determined by selection of a unique oligonucleotide sequence in the target of interest Suitable targets for splice altering MO are at an intron-exon junction in pre messenger RNA. The MO is predicted to anneal to the complimentary sequence and to inhibit intron-exon splicing. However, it is possible that an MO may anneal to an un-identified target(s) resulting in a phenotype that is unrelated to the gene of interest. Therefore it is necessary to validate MO efficacy on the targeted gene. This objective for this project is to validate anoctamin 1 splice-altering MO efficacy in zebrafish. Quantitative polymerase chain reaction (Q-PCR) will be used to compare anoctamin 1 expression levels in non-injected control embryos and in MO-injected embryos at 2 and 5 days post fertilization (dpf). Anoctamin 1 splice altering MO was designed to excise exon 4. We predict that MO knockdown will be greater at 2 dpf compared to 5 dpf. Data will be presented validating primer design and efficacy. This work will contribute to a better understanding of the general MO mechanism of action and will also validate an important technique to study anoctamin 1 function in zebrafish.

Keywords

Keywords: zebrafish, morpholino, q-PCR, quantitative PCR

Start Date

10-4-2015 9:30 AM

End Date

10-4-2015 11:00 AM

Location

Holmes 215

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Apr 10th, 9:30 AM Apr 10th, 11:00 AM

Validation of splice altering morpholino knockdown of anoctamin 1

Holmes 215

Morpholino oligonucleotides (MO) are commonly used to reduce gene expression. MO selectivity is determined by selection of a unique oligonucleotide sequence in the target of interest Suitable targets for splice altering MO are at an intron-exon junction in pre messenger RNA. The MO is predicted to anneal to the complimentary sequence and to inhibit intron-exon splicing. However, it is possible that an MO may anneal to an un-identified target(s) resulting in a phenotype that is unrelated to the gene of interest. Therefore it is necessary to validate MO efficacy on the targeted gene. This objective for this project is to validate anoctamin 1 splice-altering MO efficacy in zebrafish. Quantitative polymerase chain reaction (Q-PCR) will be used to compare anoctamin 1 expression levels in non-injected control embryos and in MO-injected embryos at 2 and 5 days post fertilization (dpf). Anoctamin 1 splice altering MO was designed to excise exon 4. We predict that MO knockdown will be greater at 2 dpf compared to 5 dpf. Data will be presented validating primer design and efficacy. This work will contribute to a better understanding of the general MO mechanism of action and will also validate an important technique to study anoctamin 1 function in zebrafish.