Development of pseudotyped retroviral system for effective gene transfer and expression in penaeid shrimp cells

Lentiviral-Transduced Ectopic Expression of Androgenic Hormone in a Crustacean Hematopoietic Primary Cell Culture

“…Indeed, we constructed a novel lentiviral vector that includes the MrIAG full mRNA sequence under WSSV IE1 promoter, which yielded a 30-40% population of positive cells compared to uninfected cells. In contrast to previous studies that evaluated fluorescent signals only microscopically (Li et al, 2011;Puthumana et al, 2015Puthumana et al, , 2016Pu et al, 2017;Shi et al, 2018;Chen et al, 2019), the quantitative data from flow cytometry analysis in our study has the advantage of offering efficient fluorescent detection and the validation of cell viability. Importantly, Mr-IAG levels in the primary hematopoietic cells were similar to those in the hypertrophied AG cells, suggesting that the MrIAG-transduced hematopoietic cells could indeed replace these cells in the first step of the biotechnology for the generation of all-female aquaculture.…”

“…The difficulties associated with the delivery of genes into crustacean cells continue to be an obstacle in the study of cellular and molecular mechanisms of crustacean species. Although considerable research using a variety of techniques has focused on the establishment of an efficient platform to insert and express a foreign gene in primary crustacean cell cultures, this research has met only with limited success, due to the difficulty of delivering foreign DNA into primary crustacean cell cultures, which enter a mitosis-arrest phase a short time after harvesting (Tapay et al, 1995;Shike et al, 2000;Lu et al, 2005;Claydon and Owens, 2008;Hu et al, 2008Hu et al, , 2010Li et al, 2011;Han et al, 2015;Puthumana et al, 2015Puthumana et al, , 2016Pu et al, 2017;Shi et al, 2018). We thus sought to leverage the ability of lentiviruses to infect both dividing and non-dividing cells by constructing a lentiviral vector that would be efficiently expressed in primary crustacean cell cultures.…”

“…Previous studies have attempted to improve gene delivery systems by using WSSV promoters to induce the expression of reporter genes, but only with limited success, possibly due to their use of an inapproriate electroporation technique for gene delivery into primary cell cultures, yielding low levels of expression of the reporter gene (Li et al, 2011;Shi et al, 2018). In the not so distant past, Pu and his colleagues attempted to overcome the tropism of pseudotyped retroviruses by the inclusion of VP19 and VP28 into the envelope of the packaged retrovirus (Pu et al, 2017), but our use of a lentivirus vector instead of a retrovirus has circumvented this need, since the successful expression of the lentiviral proteins in M. rosenbergii hematopoietic primary cell culture demonstrates high tropism of the lentiviruses to these cells. Our study suggests that the commonly used packaging lentiviral vector is capable of enhancing the expression of the desired target gene.…”

“…These tools are usually achieved by overexpression or knockdown of particular target genes, all requiring an efficient way to deliver DNA into crustacean cell cultures. A variety of techniques have been exploited for this purpose, including lipofection (Tapay et al, 1995;Claydon and Owens, 2008), electroporation (Lin and Söderhäll, 2011;Han et al, 2015;Shi et al, 2018), retroviral transduction (Shike et al, 2000;Hu et al, 2008Hu et al, , 2010Han et al, 2015;Puthumana et al, 2015;Pu et al, 2017), and baculovirus transduction (Lu et al, 2005;Puthumana et al, 2016). However, all have yielded low efficiencies of gene transfer and expression, and there remains a need for a robust system that can deliver transgenes into crustacean cells.…”

Aplikasi Bioteknologi Molekuler Dalam Budaya Perairan

“…Metode ini telah berhasil digunakan untuk membuat biota transgenik dari spesies budidaya seperti kelompok ikan catfish, ikan mas dan salmon (Dunham, 2009;Xu et al, 2011). Aplikasi metode mikroinjeksi dan elektroforesis tidak dapat dilakukan bagi biota yang perkembangan embrionya berada di dalam tubuh induknya, serta biota yang tidak melepaskan telurnya setelah pembuahan, seperti pada krustasea (udang dan lobster) (Pu et al,2017). Oleh karena itu, berkembanglah alternatif metode transfer gen yang lain, yaitu replication-defective pantropic retroviral, yaitu metode yang memanfaatkan bantuan dari sebuah vektor sebagai pembawa informasi genetik (Sarmasik et al, 2001).…”