Biomedical subjects

Isabelle Gilbert

Publications and source records attributed to Isabelle Gilbert.

2 recordsLinked to original sources

PubMed2007

Using the histone H2a transcript as an endogenous standard to study relative transcript abundance during bovine early development.

The requirement for sample standardization is basic to any relative RNA abundance assessment. In 2002, we published on the RNA abundance profiling of several housekeeping targets during early bovine embryonic development. It was then concluded that histone H2a was the most stable transcript across the studied developmental period. Since that time, several teams have applied this information, yet neglected to use the published set of primers. Here, we show that these other primer sets do not target the same histone H2a variant. Within the present report, the RNA abundance profiles of their respective targets, for example, histone H2a.1, H2a.z, and H2a.o were measured in developmental series spanning the immature oocyte to the blastocyst stage embryo. In order to more clearly define the conditions that impact the RNA abundance level measurement of these candidates, the state of polyadenylation and the origin of the transcript either from de novo transcription or from maternal stocks were taken into consideration. The histone H2a.z transcript is principally regulated by de novo transcription following embryonic genome activation, whereas the levels of H2a.1 and H2a.o variants are largely of maternal origin, conferring a more stable profile throughout the studied developmental window. The histone H2a.z was found solely in a polyadenylated state whereas the H2a.1 and H2a.o were found to be more abundant in a nonadenylated form. The presence of serum in the in vitro embryo production system also had some impact on the histone H2a.1 RNA level at the blastocyst stage.

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PubMed2006

In vivo expression of the antimicrobial defensin and lactoferrin proteins allowed by the strategic insertion of introns adequately spliced.

A major limitation of conventional shuttle expression system, when cloning a bactericidal gene, is the basal expression level in bacteria, which is lethal. Although the expression level is low, the bactericidal feature inherent to the molecule leads to subsequent failure to recover intact transformants when the related gene is cloned into a conventional expression vector. Contrary to popular belief, the human cytomegalovirus immediate-early region 1 promoter (CMV), which is to date one of the most powerful promoters for eukaryotic expression, is active in bacteria. In this study, bactericidal genes were cloned into a conventional shuttle eukaryote expression vector harbouring the CMV promoter, but were interrupted with a sequence independent splicing element (SISE), thus inhibiting lethal gene expression in bacteria. The insertion strategy of the intron uses a universal restriction site-free cloning approach, which has been developed to insert a DNA fragment into a specific location of a gene, through a PCR-based cloning technique. We have found that one intervening sequence, which derives from an adenovirus, can be spliced in a mammalian system without respect to its location, thus the bactericidal protein is synthesized only when transfected into mammalian cells. Therein, lactoferrin and defensin proteins were produced in vivo without the necessity of complex expression systems. By introducing the adeno SISE within the coding sequence of the bactericidal genes, such genes can be easily synthesized in vitro through cloning into bacteria and still are able to express biologically active proteins when introduced into mammalian cells.

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