162 EVALUATION OF THE DEMI-EMBRYOS AGGREGATION TECHNIQUE EFFICACY ON THE PRODUCTION OF CHIMERAS WITH IN VIVO PRODUCED MURINE EMBRYOS

Abstract

Mixing embryo cells coming from different fertilizations (i.e. embryonic chimera) have been used as a tool to understand embryogenesis, organo- genesis, and pluripotency, as well as a source to obtain transgenic mammals. The objectives of this work were to evaluate the potential of mice demi-embryos, in advanced stage of the development (morulae and blastocysts) to aggregate in chimeras; to compare the chimerism rate of those embryos with the rate of whole 8- to 16-cell-stage embryos; and to measure the genotype composition of the resultant chimera. One-month-old transgenic (C57/BL6/EGFP strain, GFP) or non-transgenic (Swiss Webster strain, SW) mice weighing approximately 35 g were superstimulated with 5 or 10IU of eCG (for GFP or SW mice, respectively) followed with hCG injection of 5 or 10IU (GFP or SW mice, respectively) 48 h later. Embryos were harvested at different stages of development and allocated in 3 groups for aggregation technique. Blastocysts and morulae were bisected (microblade mounted on TransferMan NK-2, Eppendorf), whereas 8- to 16-cell-stage embryos had their zona pellucida mechanically removed (23-gauge needle). Embryos were manipulated in M2 culture medium at room temperature, and aggregation groups consisted of G1 (2 demi-blastocysts, n = 28), G2 (demi-blastocyst and demi-morula, n = 20), and G3 (2 whole 8- to 16-cell-stage embryos, n = 25). All embryos were placed in wells (Embryo GPS dish, SunIVF) containing KSOMaa medium (EmbryoMax, Millipore) under oil (Sigma, St. Louis, MO, USA) and were incubated at 37°C, 5% CO₂ in air saturated with humidity. After 24 h of incubation, the presence of chimera was verified, and the percentage of area (square pixel) occupied by each embryonic type (GFP or SW) from both G2 (n = 3) and G3 (n = 3) were measured by the ImageJ program (v. 1.42i, USA). General results of the chimerism rate were 3.6%, 15.0%, and 60.0% (G1, G2, and G3, respectively; P < 0.001, chi-square). The G3 group differed from others (G1, P < 0.001 and G2, P = 0.003), which appeared similar (P = 0.294; Fisher’s exact test). The mean percentage (±SD) of GFP cells in the resultant chimera were 51.3 ± 4.1% and 50.6 ± 10.0% (for G2 and G3, respectively; P = 0.91, t-test). Moreover, the percentages of GFP cells within the same group of G2 or G3 at 0 v. 24 h of culture were not statistically different (data not shown). It was concluded that in our conditions, the embryonic chimerism by aggregation of murine demi-embryos is a feasible procedure, even for embryos in an advanced stage of development (morulae and blastocysts). Nevertheless, the chimerism rate with whole pre-compaction embryos (G3) was higher than that of G1 and G2 groups. Furthermore, the phenotype of embryonic chimera was equally composed, with no effect of strain (GFP or SW cells) or culture (0 or 24 h) on its composition.

Supported by FAPESP, Brazil: 2006/06491-2 and 2007/07705-9 (MFGN) and 2007/04291-9 (MPMM).