Production of IVF transgene- expressing embryos in bovine using a novel strategy based on cell cycle inhibitors

ROMINA BEVACQUA; PEREYRA-BONNET FEDERICO; OLIVERA RAMIRO; HIRIART MI; SIPOWICZ PABLO; FERNANDEZ-MARTIN RAFAEL; RADRIZZANI MARTIN; SALAMONE DANIEL

THERIOGENOLOGY

ELSEVIER SCIENCE INC

Lugar: Amsterdam; Año: 2012 vol. 78 p. 57 - 68

0093-691X

AbstractThe objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) ontransgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-enhanced green fluorescent protein (EGFP) gene plasmid (50 ng/L) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mM DMAP or 1 M DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v DMAP, leDNA-v, and leDNA, respectively) and also increased (P > 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P > 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P >0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v  DMAP, leDNA-v  DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration inegfp embryos. Finally, clones derived from leDNA-v  DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v DMAP, leDNA-v, and leDNA, respectively).