Methods and Compositions for Producing Active Vitamin K-Dependent Proteins
a technology of vitamin k and protein, applied in the field of isolating nucleic acids and host cells, can solve the problems of significant risk of side effects, accompanied by warfarin utilization, mental retardation and facial abnormalities, etc., and achieve the effects of increasing or decreasing warfarin sensitivity
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example i
Correlation Between SNPs in VKOR Gene and Increased or Decreased Sensitivity to Warfarin
[0088]The most prevalent isoform of the VKOR gene is about 4 kb long, has three exons and encodes an enzyme of 163 amino acids with a mass of 18.4 kDa. In the present study, three mutations vk2581 (G>C), vk3294(T>C) and vk4769(G>A), identified as SNPs (heterozygosity ratios of 46.9%, 46.8% and 46.3%, respectively) were examined for a correlation between their presence in a subject and the maintenance dose of warfarin required to achieve a therapeutically effective response.
1. Selection of Subjects
[0089]Subjects were obtained from the UNC Coagulation Clinic in the Ambulatory Care Center. Informed consent was obtained by a trained genetic counselor. Subjects not fluent in English were excluded because of the lack of translators and the requirement for consent. To qualify for the study, subjects had warfarin for at least six months, were older than 18 and were followed by the UNC Coagulation clinic ...
example 2
siRNA Design and Synthesis
[0100]siRNAs were selected using an advanced version of a rational design algorithm (Reynolds et al. (2004) “Rational siRNA design for RNA interference”Nature Biotechnology 22:326-330). For each of the 13 genes, four siRNAs duplexes with the highest scores were selected and a BLAST search was conducted using the Human EST database. To minimize the potential for off-target silencing effects, only those sequence targets with more than three mismatches against un-related sequences were selected (Jackson et al. (2003) “Expression profiling reveals off-target gene regulation by RNAi”Nat Biotechnol 21:635-7). All duplexes were synthesized in Dharmacon (Lafayette, Colo.) as 21-mers with UU overhangs using a modified method of 2′-ACE chemistry (Scaringe (2000) “Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis”Methods Enzymol 317:3-18) and the AS strand was chemically phosphorylated to ensure maximum activity (Martinez et al. (2002) “Single-...
example 3
siRNA Transfection
[0101]Transfection was essentially as previously described (Harborth et al. (2001) “Identification of essential genes in cultured mammalian cells using small interfering RNAs”J Cell Sci 114:4557-65) with minor modifications.
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