Koo-Jeong Kang | PubMed Health

PubMed2005

Gene knockdown by large circular antisense for high-throughput functional genomics.

Single-stranded genomic DNA of recombinant M13 phages was tested as an antisense molecule and examined for its usefulness in high-throughput functional genomics. cDNA fragments of various genes (TNF-alpha, c-myc, c-myb, cdk2 and cdk4) were independently cloned into phagemid vectors. Using the life cycle of M13 bacteriophages, large circular (LC)-molecules, antisense to their respective genes, were prepared from the culture supernatant of bacterial transformants. LC-antisense molecules exhibited enhanced stability, target specificity and no need for target-site searches. High-throughput functional genomics was then attempted with an LC-antisense library, which was generated by using a phagemid vector that incorporated a unidirectional subtracted cDNA library derived from liver cancer tissue. We identified 56 genes involved in the growth of these cells. These results indicate that an antisense sequence as a part of single-stranded LC-genomic DNA of recombinant M13 phages exhibits effective antisense activity, and may have potential for high-throughput functional genomics.

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PubMed2004

Optimal cycle of intermittent portal triad clamping during liver resection in the murine liver.

We designed this experimental study to determine the optimal cycle for intermittent inflow occlusion during liver resection. A cycle of intermittent clamping (IC) for 15 minutes of ischemia followed by reperfusion for 5 minutes during liver resection is currently the most popular protocol used by experienced liver centers. As each period of reperfusion is associated with bleeding, longer periods of clamping would be advantageous. However, the longest safe duration of successive ischemia is unknown. Three groups of mice were subjected to a total liver ischemic period for 90 minutes; 2 groups underwent IC for 15 or 30 minutes, respectively, followed by 5 minutes of reperfusion, while the control group was subjected to continuous inflow occlusion only. The degree of tissue injury was assessed using biochemical and histological markers, as well as animal survival. While serious injury was observed in the continuous clamping group, both IC groups were associated with minimal injury, including lesser degrees of apoptosis and necrosis. All animals survived in the IC groups, while all animals died following 90 minutes of continuous inflow occlusion. In conclusion, intermittent portal pedicle clamping with 15- or 30-minute cycles is highly protective. A period of 30 minutes clamping should be preferred, since this would decrease the amount of blood loss associated with each cycle. This data should be confirmed in humans, and may represent a change in the current practice of hepatic surgery.

Animals↗