289 DERIVATION OF MOUSE EMBRYONIC STEM CELL FROM C57BL/6/EGFP STRAIN WITH FETAL CALF SERUM AND KNOCKOUT SERUM REPLACEMENT® SUPPLEMENTATION MEDIUM

Embryonic stem cells (ESC) have been used in attempts to obtain specific tissues or even individuals. Embryonic stem cells are pluripotent, allowing the differentiation of cell types from 3 germ layers. The establishment of a stable lineage of ESC is a valuable tool; however, some strains of mice are less permissive to ESC derivation or generation of chimeric animals (e.g. C57BL/6). Supplementation of culture medium with FCS, in the ESC derivation, may influence the potentiality to derivation or use of these strains in tetraploid complementation assays (Sato et al. 2009 Tsukuba Res. Inst. 47, 414–422). Thus, its replacement was carried out using knockout serum replacement (KSR®) to minimize the deleterious action of serum (Wang et al. 2007 Inst. of Biotech. 23, 269–272). Embryos were obtained from 5 females of lineage C57BL6/EGFP, aged between 21 and 30 days and weighing ∼35 g, and superstimulated according (Mancini et al. 2008 Transg. Res. 17, 1015). The animals were placed for mating with fertile males of the same strain in a proportion of one to one (male:female). The copulation was confirmed by plug vaginal (0.5 days postcopulation). Embryo recovery was performed 3.5 to 4.0 days postcopulation to obtain expanded (EB) or hatched blastocysts (HB). Zona pellucida was removed from EB with the aid of pronase solution, and the whole embryos (n = 8) were placed on a 4-well dish pretreated with pig skin gelatin 0.1%, under murine fibroblast primary in DMEM medium supplemented with 7.5% FCS and 7.5% KSR®, 10 mM βmercaptoetanol, 1 mM sodium pyruvate, 2 mM L-glutamine, and 83.4 mg mL–1 amikacin for 24 h. After this period, the medium was replaced by DMEM supplemented with 15% KSR®. The colonies began to grow between 3 and 6 days after in vitro culture of the embryos. Once established, the colony was picked and placed into new plates containing murine fibroblast primary every 48 to 72 h. After 14 days, the derivation was confirmed with some proved pluripotency markers by immunofluorescence (Oct3/4, SSEA-1, and Nanog) and karyotyping for ploidy detection. The reaction was positive for all tested markers in addition to the detection of the endogenous fluorescence from EGFP protein itself (C57BL/6EGFP origin). It was concluded that ESC derivation with partial serum replacement and using a less permissive strain such as C57BL/6EGFP is feasible, although with a reduced success rate (12.5%; i.e. 1 lineage – named BCM04 – from 8 attempts). Fellowships and grants were received from FAPESP, Brazil: 09/15919-4 (BCSC), 09/16254-6 (DMS), 09/17605-7 (CPG), 06/06491-2 (MFGN), and 07/07705-9 (MFGN).