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English words and Examples of Usage
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Sentences for "antisense"
We have identified cells expressing IDO in vitro and used IDO antisense constructs to inhibit this expression.
Inhibition of endogenous IDO expression disrupts P19 cell adhesion To examine the role of IDO expression in P19 cells, we transfected P19 cells with a DNA construct which contained a 740 bp fragment of murine IDO cDNA in the antisense orientation, under the control of the constitutively active CMV promoter in the pcDNA-3 mammalian expression vector.
To confirm that transfected, G418 resistant P19 cells expressed IDO antisense transcripts, we isolated total RNA from G418 resistant clones, reverse transcribed it into cDNA using a sense primer then PCR amplified the cDNA.
Three antisense-transfected clones, which expressed progressively greater amounts of IDO antisense RNA were selected for further analysis together with a sense control (Fig 4D).
The ability of the sense and antisense transfectants to deplete tryptophan from culture medium was determined following 48 hours in culture.
IDO sense transfected P19 cells depleted 10% of available culture tryptophan (not shown) while IDO antisense transfected P19 clones which expressed high levels of antisense (clones D3 and E6) depleted essentially no tryptophan from the medium.
IDO antisense expressing clones developed a rounded appearance with a more scattered morphology and apparent loss of cell interaction instead of the usual, adherent P19 phenotype.
The degree to which this phenotype manifested, correlated with the extent of IDO antisense expression, i.
5F) produced aggregates which were only loosely packed and a substantial number of cells which did not package into any form of aggregate while antisense clone D3 formed aggregates more diverse in shape than the uniformly spherical controls but less diverse than clone E6.
To quantitate and compare the size difference between sense and antisense-transfected aggregates we photographed sense and antisense clone 5 aggregates and calculated the area of each aggregate individually (Fig.
When inserts were coated with Matrigel, no significant migration was seen in either antisense or sense transfectants, indicating that cell motility could be inhibited by supplying an extracellular matrix.
Furthermore, there was a progressive increase in the expression level of these three protease genes, which correlated with the amount of IDO antisense expression.
P19 cells in which endogenousIDO expression was disrupted by antisense expression, showed lower adhesiveness.
Antisense inhibition of IDO expression Constructs which expressed either sense or antisense IDO RNA were produced by cloning a 740 bp RT-PCR fragment of the IDO gene, described in [ 17], into the T-tailed cloning vector pGEM T-Easy (Promega).
This fragment was excised with Not I and subcloned into the Not I site of the mammalian expression vector pcDNA3 (Invitrogen), in either the sense or antisense orientation.
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