Effect of high hydrostatic pressure on the in vitro development and molecular quality of transgenic rabbit embryos derived from nano-transfected zygotes

Abstract

The aim of this study was to evaluate the effect of high hydrostatic pressure (HHP) on the in vitro developmental abilities of nano-transfected rabbit zygotes, their transfection efficiency, and the molecular quality of the blastocysts generated. This quality was assessed by estimating the quantitative profiles of Oct4, Casp7, and Bcl2 mRNA transcripts. The nano-transfection efficiencies of zygotes that had been pre-treated with either 20 MPa or 40 MPa of HHP (13.5% and 13.7%, respectively) were insignificantly lower than those found in zygotes not exposed to HHP prior to their nano-transfection (20.1%; P≥0.05). Moreover, applying HHP treatment with the parameters of 20 MPa and 40 MPa followed by the nano-transfection of zygotes brought about an insignificant decrease in the rates of embryos at the blastocyst stage (30.4% and 23.0%, respectively) as compared to the control group of nano-transfected zygotes (40.4%; P≥0.05). Furthermore, analyzing the transcriptional activity of Oct4, Bcl2, and Casp7 genes revealed that HHP enhances the relative abundance (RA) of all mRNA transcripts in blastocysts derived from non-transfected rabbit zygotes. In turn, the augmented RAs found in the pro-apoptotic Casp7 and anti-apoptotic Bcl-2 transcripts confirmed the onset and progression of programmed cell death in blastocysts developed from nano-transfected zygotes that had undergone HHP pre-treatment. The conceptualization based not only on a novel nano-transfection approach used to genetically modify in vivo-fertilized rabbit zygotes but also on their HHP pre-treatment is elaborated here for the first time, with an emphasis on further investigations aimed at producing transgenic rabbit and other mammalian species embryos by somatic cell cloning.

The applicability of nanobiotechnology-related approaches to veterinary medicine and assisted animal reproduction – A review

“…The selection of cell types for nanotransfection is often determined by the purpose of application, e.g., gene editing, gene addiction, or gene inhibition. As nanotechnology-based approaches to assisted animal reproduction mainly concern gene editing, two different strategies are usually taken into account: the nanofection of somatic cell primary cultures or clonal lines (for the purpose of somatic cell nuclear transfer; SCNT) or the nanoparticle-mediated transgenization of zygotes (via intrapronuclear microinjection) (Grześkowiak et al, 2015;Dzięgiel et al, 2022).…”

“…The above-indicated strategies for the nanoparticle-assisted transfection of in vitro cultured somatic cells, in a variety of mammalian species, may be reliable and feasible for propagating and multiplying the abundant sources of genetically engineered or genome-edited nuclear donor cells for the purposes of cloning via SCNT technique (Perisse et al, 2021;Skrzyszowska and Samiec, 2021;Samiec, 2022;Samiec et al, 2022). Finally, nanoparticle-mediated gene delivery seems to be also a promising tool for the transgenization of in vivo fertilized rabbit zygotes by their intrapronuclear microinjection with pmaxFP-Red-N (eRFP-based) gene constructs; this might also open up new possibilities for the nanotransfection of cloned embryos at the early 1-blastomere stages that are generated in the post artificial activation of SCNTderived oocytes (Dzięgiel et al, 2022).…”