Abstract Number: PB0139
Meeting: ISTH 2020 Congress
Background: The piggyback knockdown (PK) method was used to study hemostasis by performing genome-wide screens using zebrafish. Here, we intravenously inject a vivo morpholino (VMO) that piggybacks an antisense deoxyoligonucleotide into the somatic cells and somehow reduces mRNA levels. The application of the PK method requires enhancing efficiency and understanding the mechanism.
Aims: To increase efficiency and understand the mechanism of the PK method.
Methods: PKs, kPT, kPTT assays, qRT-PCRs, and thrombocyte aggregation assay were performed according to published methods. Student’s t-test was used in statistical analysis.
Results: To increase the efficiency of the PK method, we tested whether we can piggyback more than one deoxyoligonucleotide with one VMO. We designed hybrids that had more than one deoxyoligonucleotide that could be piggybacked with VMO. Since we have only 25 bases in the VMO, tested 10, 7, and 5 bp lengths to accommodate pairing of 2, 3, and 4 different deoxyoligonucleotides. We used f7, f8, and αIIb and tested the efficiency of knockdown by kPT, kPTT, and thrombocyte aggregation assay, respectively. The PK using four deoxyoligonucleotides did not yield acceptable efficiency, whereas the knockdown with two and three deoxyoligonucleotides yielded excellent results. We used qRT-PCR to check the efficiency of knockdown and found that all three genes were knocked down by >85%. As expected, we also noted bleeding spots in 20-30% of these fish. We also performed knockdown of RNaseH and argonautes along with f7 and found the knockdown occurs with argonautes and not with RNaseH suggesting that RNaseH is involved in mRNA degradation.
Conclusions: We developed a method where we could knockdown three genes at one time. We established that knockdown is due to RNaseH-mediated mRNA degradation. Multiple gene knockdowns (up to 6 genes) will not only increase the efficiency of the method in genome-wide knockdowns but will also be useful to study multifactorial disorders.
To cite this abstract in AMA style:Raman R, Ryon M, Jagadeeswaran P. Multiple Gene Knockdowns Simultaneously to Study Hemostasis in Zebrafish [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/multiple-gene-knockdowns-simultaneously-to-study-hemostasis-in-zebrafish/. Accessed November 29, 2023.
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