Use of a Sendai virus-based vector for effcient transduction of pinniped fbroblasts

Abstract

Generation of induced pluripotent stem (iPS) cells expanded possibilities of pluripotency and early development studies. Generation of order Carnivora iPS cells from dog (Canis lupus familiaris), snow leopard (Panthera uncia), and American mink (Neovison vison) was previously reported. The aim of the current study was to examine conditions of pinniped fbroblast reprogramming. Pinnipeds are representatives of the suborder Caniformia sharing conservative genomes. There are several ways to deliver reprogramming transcription factors: RNA, proteins, plasmids, viral vectors etc. The most effective delivery systems for mouse and human cells are based on viral vectors. We compared a lentiviral vector which integrates into the genome and a Sendai virus­based vector, CytoTune EmGFP Sendai Fluorescence Reporter. The main advantage of Sendai virus­based vectors is that they do not integrate into the genome. We performed delivery of genetic constructions carrying fluorescent proteins to fbroblasts of seven Pinnipeds: northern fur seal (Callorhinus ursinus), Steller sea lion (Eumetopias jubatus), walrus (Odobenus rosmarus), bearded seal (Erignathus barbatus), Baikal seal (Pusa sibirica), ringed seal (Phoca hispida), and spotted seal (Phoca largha). We also transduced American mink (N. vison), human (Homo sapiens), and mouse (Mus musculus) fbroblasts as a control. We showed that the Sendai virus­based transduction system provides transgene expression one­two orders of magnitude higher than the lentiviral system at a comparable multiplicity of infection. Also, transgene expression after Sendai virus­based transduction is quite stable and changes only slightly at day four compared to day two. These data allow us to suggest that Sendai virus­based vectors are preferable for generation of Pinniped iPS cells.

Derivation of Ringed Seal (Phoca hispida) Induced Multipotent Stem Cells

“…Another way to increase efficiency is to use different transgenes and/or delivery vectors. We have recently shown that Sendai virusbased vector transgene expression level is superior to lentiviruses (Beklemisheva, Menzorov, 2018). Also, another set of reprogramming factors was successfully used to reprogram not only human, but ferret fibroblasts to pluripotency, OCT4, SOX2, KLF4, L-MYC, LIN28A, mp53DD, and 160 oriP/EBNA-1 (Gao et al, 2020).…”

“…Lentiviruses were produced in the Phoenix cell line using Lipofectamine 3000 (Thermo Fisher Scientific, USA). The multiplicity of infection was estimated as 5.1 using EGFP lentiviral vector (Beklemisheva, Menzorov, 2018). Fibroblasts at passage 4 (3 x 10 5 cells, 30 x 10 3 cells/cm 2 ) plated the day before were transduced with viruses containing four reprogramming transcription factors: 50 % virus supernatant, 50 % fibroblast culture medium without antibiotics with heat-inactivated FBS, and 10 µg/ml Polybrene.…”

Derivation of ringed seal (Phoca hispida) induced multipotent stem cells

“…Another way to increase efficiency is to use different transgenes and/or delivery vectors. We have recently shown that Sendai virus-based vector transgene expression level is superior to lentiviruses (Beklemisheva, Menzorov, 2018). Also, another set of reprogramming factors was successfully used to reprogram not only human, but ferret fibroblasts to pluripotency, OCT4, SOX2, KLF4, L-MYC, LIN28A, mp53DD, and 160 oriP/EBNA-1 (Gao et al, 2020).…”

“…Lentiviruses were produced in the Phoenix cell line using Lipofectamine 3000 (Thermo Fisher Scientific, USA). The multiplicity of infection was estimated as 5.1 using EGFP lentiviral vector (Beklemisheva, Menzorov, 2018). Fibroblasts at passage 4 (3 × 10 5 cells, 30 × 10 3 cells/cm 2 ) plated the day before were transduced with viruses containing four reprogramming transcription factors: 50 % virus supernatant, 50 % fibroblast culture medium without antibiotics with heat-inactivated FBS, and 10 µg/ml Polybrene.…”

Pluripotent Stem Cells of Order Carnivora: Technical Perspective

“…The risk of genome integration of DNA-based vectors exists but overall is negligible. We have successfully tested Sendai-virus-based CytoTune EmGFP Sendai Fluorescence Reporter (Thermo Fisher Scientific, Carlsbad, CA, USA) in pinniped fibroblasts [ 47 ]. On day 4, the fluorescence was significantly higher for the Sendai-virus-based vector than for the lentiviral-based one.…”