72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN PERIVITELLINE SPACE MICROINJECTION

Abstract

The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the perivitelline space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO₂ and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components.

Financial support was received from FAPESP of Brazil.