Other vectors contain att recombination sequences exterior to the TOPO cloning sites so that cloned inserts are ready for entry into the Gateway system. Many Invitrogen expression vectors are adapted for one-step TOPO Cloning of PCR products in both directional and non-directional formats. After 5 minutes at room temperature, enzyme is released, the ligation is complete and the recombinant molecule is ready for transformation into E. This enables fast ligation of DNA sequences with compatible ends. To har- a ness this activity, vectors are linearized and each end is conjugated with topoisomerase on the 3' phosphate. The key to TOPO Cloning is the enzyme DNA topoisomerase I, whose biological role is to cleave and rejoin DNA during replication. TOPO Technology is a fast, efficient way to clone. The second step transfers the sequence into a variety of attB-containing Expression Clones that can be propagated and expressed in a range of host cells for a given experiment. In overview, Gateway is a 2-step cloning process in the first step a sequence of interest is inserted into an Entry Clone. This system is based on the well-characterized bacteriophage lambda-based sitespecific recombination system (attL x attR ↔ attB x attP). 86(8):4527-37.Introduction to Gateway Cloning C 23 HAPTER ® GATEWAY AND TOPO CLONING Gateway Cloning Technology is Invitrogen’s universal cloning system that provides a rapid and highly efficient route to protein expression, functional analysis and cloning/subcloning of DNA segments. Sensing adenovirus infection: activation of interferon regulatory factor 3 in RAW 264.7 cells. Recognition of nucleic acids by pattern-recognition receptors and its relevance in autoimmunity. IFNbeta responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI). The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. IFI16 is an innate immune sensor for intracellular DNA. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. CpG motifs in bacterial DNA trigger direct B-cell activation. The RAW 264.7 macrophages have been reported to express several CDSs IFI16, DDX41 and AIM29.These cells express a reporter gene, either SEAP (secreted embryonic alkaline phosphatase) or Lucia, a secreted luciferase, under the control of an IRF-inducible promoter.ġ. The human monocytic cell line THP-1 has been shown to express all the CDSs, with the exception of DAI. In order to facilitate their study, InvivoGen has developed stable reporter cells in two well established immune cell models, the human monocytic THP-1 cell line and the murine RAW 264.7 macrophages. CDS ligands, including transfected dsDNA-EC, trigger type I interferon (IFN) production and the induction of IFN-stimulated genes (ISG) through interferon regulatory factors (IRFs). Unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs), present on bacterial DNA, are recognized by TLR9 which subsequently engages an intracellular pathway leading to NF-κB activation. Intracellular DNA is recognized by the innate immune system via the endosomal TLR9 receptor and multiple cytosolic DNA sensors (CDSs) which display contextual preferences for the recognition of DNA.
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