lly recombined ones. Higher get Cy5 NHS Ester expression was confirmed by the use of another couple of primers which amplifies both endogenous and transgenic TIAR mRNAs. Correlatively, higher levels of transgenic TIAR protein were detected in TIAR-GFPDp testis than in TIAR-GFPDt testis. These data indicate that partial excision of GFP cassettes leads to higher expression levels of transgenic TIAR protein than their complete deletion. When applied to embryonic development, this reasoning implicates that embryos containing partially excised transgenes would express higher levels of transgenic TIAR protein than those containing only one excised copy of the transgene, the latter being able to fully develop while the former would die. Impaired development of TIAR overexpressing embryos 
at post-implantation stages To test this hypothesis, we compared the development of embryos resulting from the mating of heterozygous GFP-TIAR transgenic males with WT or PGK-cre females. Embryos were collected between TIAR-Overexpressing Embryos number of developmentally delayed or abnormal embryos with age. To know whether retarded or degenerating embryos were transgenic, we either cultivated the ectoplacental 18325633 cone of E Increased sensitivity of PGK-Cre x GFP-TIAR blastocysts to in vitro culture pre-implantation stages resulting in embryonic lethality shortly after implantation. To verify this hypothesis, we first analysed the stage at which TIAR accumulation could start taking first GFP as a reporter of b-actin promoter activity in embryos recovered from WT x GFP-TIAR crosses. GFP expression was detectable from 11325787 the morula stage and beyond. Thus, after Cre recombination, the transgenic TIAR protein is not expected to be expressed before morula stage. Unfortunately, we could not analyse specifically TIAR transgenic expression due to high background signal when using anti-FLAG antibody. We therefore determined whether TIAR was overexpressed in transgenic embryos by immunofluorescence analysis of in utero collected blastocysts from WT or PGK-Cre females mated with homozygous GFP-TIAR. We observed by confocal microscopy that PGK-Cre-derived blastocysts were indeed more stained than the WT ones with a strong staining of the nucleus of each blastomere. Such TIAR overexpression has no apparent detrimental consequences on in utero pre-implantation development since healthy blastocysts were recovered in utero with the same yield from WT or PGK-Cre females mated with homozygous GFP-TIAR a and b males. Then, we analysed the ability of TIAR overexpressing embryos to develop in vitro. Fertilized one-cell stages or morula recovered from WT or PGK-Cre females were June TIAR-Overexpressing Embryos June TIAR-Overexpressing Embryos June TIAR-Overexpressing Embryos cultured in M Discussion named mid-preimplantation gene activation, that precedes the dynamic morphological and functional changes from the morula to blastocyst stage. At the protein level, our confocal analysis revealed that TIAR protein is expressed during pre-implantation stages and that, due to transgene expression, TIAR is more expressed in -derived blastocysts than in WT or age-matched control ones. Such TIAR accumulation, mainly in the nucleus, did not apparently perturb in utero pre-implantation development since healthy blastocysts were recovered with the same yield from WT or PGK-Cre females. However, TIAR blastocyst formation was compromised in the sub-optimal MJune TIAR-Overexpressing Embryos any obvious difference between
