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Expression of tagged glp-1 alleles was first reported for a C-terminal Dendra2::GLP-1 fusion protein expressed in a transgenic strain (Di Orio et al., 2015). This fusion protein is made at endogenous levels and is predicted to be membrane bound, because its last extracellular and transmembrane domains are deleted (Cinquin et al., 2015). Two additional transgenes, also expressed from the endogenous locus, were created by inserting the Dendra2::GLP-1 C-terminal tag into the endogenous glp-1 locus (Figure 1). One of these is a transgene that carries a deletion of the glp-1 intracellular domain and thus has constitutive activity (gpn-1::Dendra2::GLP-1, Figure 1A). The other is a CRISPR tag that was inserted into the glp-1 locus, resulting in a frame-shift mutation that deletes the last 24 amino acids of glp-1 and introduces a stop codon that also fuses the Dendra2 tag to the C-terminus of the remaining protein (Figure 1B). Both transgenes are expressed ubiquitously (Figure 2), but Dendra2::GLP-1 is not ubiquitously expressed (Figure 2), suggesting that this fusion protein has a subcellular localization pattern distinct from GLP-1. Whether the localization of the Dendra2::GLP-1 fusion protein reflects the natural distribution of the glp-1 receptor or is due to the fusion construct remains to be determined.
The NICD antibody used in previous studies is not specific for Notch activation, and lacks the ability to distinguish between pro-Notch and activated Notch. We therefore also generated three CRISPR mutants that contain alleles of the glp-1 locus with endogenous glp-1 tagged with (1) GFP, (2) V5 epitope tag, and (3) yellow fluorescent protein (YFP). These strains are suitable for examining glp-1 activation using the NICD antibody (Figure 1).
We report here that we tagged the endogenous glp-1 locus in three distinct ways. To do this, we: (1) introduced transgenes encoding GFP-tagged glp-1 (pGLP-1::GFP) and glp-1 fused to enhanced YFP (pGLP-1::YFP), (2) created a single nucleotide deletion using CRISPR/Cas9 at the endogenous glp-1 locus, and (3) introduced a multi-copy version of a CRISPR/Cas9-targeted gfp-tagged glp-1 into the single nucleotide deletion mutant.
Twidosuite is a powerful and easy-to-use program that will allow you to control your Twidosuite based programmable controller from your PC. You can get a visual representation of your controller and program settings on your PC. The program is very easy to use and will let you get started with your controller without spending a lot of time in the software. It also provides the ability to download new firmware, which is required for the most recent controllers you may have. This new firmware also provides a lot of nice new features including the ability to download and burn new circuits to a new controller. There are also a lot of new features and enhancements to the program as well.
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