Energy aware sensor management for wearable medical systems optimization
a sensor management and wearable technology, applied in the field of energy aware sensor management for wearable medical systems, can solve the problems of difficult to achieve vaccination of all individuals, less successful approaches to tackle many viruses of significant medical and/or veterinary importance, and major causes of disease and death
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Characterization and Retroviral Infection of KBM7 Subclones
[0184]We first characterized a haploid genome setting in human cells that we believed would be permissive for efficient forward genetic approaches. A subclone of the CML cell line KBM7 has been described to carry a near haploid chromosome set (Kotecki, M., Reddy, P. S., and Cochran, B. H. Isolation and characterization of a near-haploid human cell line. Exp Cell Res 252, 273-280, 1999). First we examined if this cell line (generously provided by Dr. B. H. Cochran, Tufts University School of Medicine, Boston, Mass.) could be easily propagated, was tolerant to viral infection and could be efficiently subcloned. The term “KBM7 cell line” is used herein to refer to this near-haploid cell line or to a subclone thereof. Cells of the KBM7 cell line or a subclone thereof may be referred to as “KBM7 cells”. KBM7 cells had a high subcloning efficiency (of around ˜80%), and several of the subclones were examined further. The KBM7 subcl...
example 2
Retroviral Infection of KBM7 Cells
[0185]We next showed that KBM-7 cells could be infected with retroviruses. Virus was produced by transfection of a GFP expressing retroviral vector with packaging vectors in 293T cells (obtained from ATCC). The retroviral vector was pLIB-GFP (Clontech) but it will be understood that many different retroviral vectors could be used. Supernatant containing virus was used to infect KBM7 cells. To improve the infection efficiency of KBM7 cells with retroviruses, different conditions were tested. Centrifugation of the cells in a 24-well tissue culture dish for 45 minutes at 2,000 pm at room temperature resulted in a 2-fold increase in infection efficiency compared to no centrifugation. Next the effect of retronectin, polybrene and protamine sulphate addition was tested, yielding efficiencies of 25%, 33% and 44%, respectively. Eight microgram per milliliter culture medium of protamine sulphate is the preferred addition. Concentration of virus by ultracentr...
example 3
Construction of Gene Trap Vectors Containing Vectors Containing Puromycin and GFP Selectable Markers
[0186]Retroviral gene trap vectors that contain an inactivated LTR, a strong splice-acceptor site derived from the long fiber gene of Adenovirus serotype 40 (Carette et al. 2005 The Journal of Gene Medicine 7(8) 1053-1062), and either GFP or the puromycin resistance gene (PURO) followed by a SV40 polyadenylation signal were constructed as follows. The coding sequence of the PURO or GFP was obtained by PCR amplification with primers containing overhanging ClaI and NheI restriction sites as well as partial splice acceptor sites: (GFP:5′-GATCGCTAGCCGCATTTCTTTTTTCCAGATGGTGAGCAAGGGCGAGG-3′ and 5′-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3′ PURO: 5′-GATCGCTAGCCGCATTTCTTTTTTCCAGATGACCGAGTACAAGCCCAC-3′ and 5′-GATCGGATCCTCAGGCACCGGGCTTGCGGGTC-3′). These PCR products were inserted in pEGFPC1 (Clontech) replacing EGFP. Subsequently PCR was performed to introduce the complete splice acceptor site and to...
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