Vitrification of pre-implantation embryos a simple method for the preservation and production of transgenic mice
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Abstract
Vitrification of embryos and gametes is a novel alternative for ex situ preservation and production of transgenic mice. The first objective of this research was to demonstrate the effectiveness of vitrifying 4-cell embryos for the preservation of genetic lines from four transgenic mouse models: 1) Ataxia, 2) Human transferrin, 3) CD46 receptor, and 4) transgenic mice expressing green fluorescent protein (GFP). A second objective was to produce transgenic offspring from vitrified pronuclear embryos that had been genetically modified via lentiviral transgenesis. The embryos obtained from non-transgenic mice served as the control group. Those embryos at the 4-cell and pronuclear stages were collected 55 hours and 18 hours, respectively, after superovulatory treatment. The embryos collected were vitrified using specific mixtures of DMSO, ethylene glycol and sucrose; and they were subsequently stored in liquid nitrogen until needed. In vitro and in vivo survival was evaluated in each experimental group, as well as the principal cytostructural damage suffered by the embryos after thawing. In the first experiment, a high embryonic survival rate was reached in all four transgenic lines after the vitrification of embryos at the 4-cell stage, ranging from 91,5 % to 100 %. In the case of in vivo development, no significant differences were observed in the percentages of viable offspring born among the transgenic lines Ataxia (49,5 %), Transferrin (47 %) and CD46 receptor (24,4 %), compared to the vitrified control group (38,3 %) (p>0,05). The main cytostructural damages observed were the rupture of the zona pellucida and lysis of blastomeres, with percentages differing among groups. In the case of pronuclear embryos, a 19,9 % viable offspring rate was obtained (n=52 live births/266 embryos transferred). Of these, 25 % (n=13/52) of the offspring obtained were transgenic, resulting in an overall efficiency of 4,5 % (n=13/266) for lentiviral transgenesis using vitrified pronuclear embryos. The results obtained demonstrate that embryo vitrification is an effective method for preserving mouse lines and producing transgenic mice through lentiviral transgenesis.
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