Morpholino Oligomer Delivery by Bath Immersion Enables Reverse Genetic Studies in the Early Development of Eastern Oysters (Crassostrea Virginica)

17 Pages Posted: 14 May 2024

See all articles by Lan Xu

Lan Xu

affiliation not provided to SSRN

Jessica Moss Small

affiliation not provided to SSRN

Shannon M. Hood

affiliation not provided to SSRN

Mingli Zhao

affiliation not provided to SSRN

Louis V. Plough

affiliation not provided to SSRN

Ten-Tsao Wong

affiliation not provided to SSRN

Abstract

The eastern oyster genome and expanding omics data have provided valuable insights into this species. However, the limited availability of molecular toolboxes constrains the further exploration of gene functional investigation. In this study, we applied an emerging immersion-based gene silencing technology and developed a detailed protocol for delivering morpholino oligomer (MO) to eastern oysters. First, two target genes, cv-gcl and cv-vasa were cloned, and their expressions in ovaries and during embryogenesis were characterized. Both genes were maternally deposited to oocytes, and the expression of cv-vasa decreased steadily after fertilization, while cv-gcl peaked around 20 hours post-fertilization. The post-fertilization immersion treatment was more effective (three- to four-fold) than pre-fertilization treatment, indicating that eastern oysters may have a more active MO uptake mechanism after fertilization. MOs designed against these two genes were administered via Vivo using bath immersion treatment following fertilization. There was no difference in survival rates (D-larvae yield) at 1 day post-fertilization (dpf) when treating embryos with cvgcl-MO-Vivo up to 20 µM, while deleterious effects started to emerge when increasing the concentration to 30 and 40 µM. The cvvasa-MO-Vivo treated group exhibited a more than six-fold reduction on the Vasa level, suggesting the effectiveness of immersion treatment and the knockdown of the target gene. Most importantly, we achieved real-time visualization of MO uptake by conjugating fluorescence-labeled MO with a cell-penetrating peptide. Monitoring fluorescent intensity inside oyster larvae revealed that MO delivery occurred during early embryogenesis, and the signal retained up to 6 dpf. The immersion and fluorescence-traceable approach described here is a highly efficient reverse genetic method of delivering and monitoring MO in a large number of developing eastern oyster embryos to study the function of genes of interest involved in early development.

Keywords: MO-Vivo, knockdown, embryo development, vasa, germ-cell less

Suggested Citation

Xu, Lan and Small, Jessica Moss and Hood, Shannon M. and Zhao, Mingli and Plough, Louis V. and Wong, Ten-Tsao, Morpholino Oligomer Delivery by Bath Immersion Enables Reverse Genetic Studies in the Early Development of Eastern Oysters (Crassostrea Virginica). Available at SSRN: https://ssrn.com/abstract=4827134 or http://dx.doi.org/10.2139/ssrn.4827134

Lan Xu

affiliation not provided to SSRN ( email )

No Address Available

Jessica Moss Small

affiliation not provided to SSRN ( email )

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Shannon M. Hood

affiliation not provided to SSRN ( email )

No Address Available

Mingli Zhao

affiliation not provided to SSRN ( email )

No Address Available

Louis V. Plough

affiliation not provided to SSRN ( email )

No Address Available

Ten-Tsao Wong (Contact Author)

affiliation not provided to SSRN ( email )

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