Proinsulin Tracking System Generation

ProCpepRUSH (SA-KDEL-IRES-proCpep-SBP-GFP) was generated by Gibson assembly and subcloned into pENTR2b-RIP^{12 (link)}. SA-KDEL-IRES and SBP PCR fragments were separately amplified from Str-KDEL_SBP-EGFP-GPI (gift from Franck Perez, Addgene plasmid #65,294; RRID:Addgene_65294)^{28 (link)}. sfGFP was PCR amplified from sfGFP-C1 (gift from Michael Davidson and Geoffrey Waldo, Addgene plasmid #54,579; RRID:Addgene_54579)^{65 (link)}. Proinsulin was PCR amplified as two fragments on either side of the ApaI site from human INS cDNA for insertion of the SBP and sfGFP fragments within the C-peptide coding sequence. RIP-SA-KDEL-IRES-proCpep-SBP-GFP was recombined from pENTR2b-RIP into a modified pAD-PL/DEST via Gateway cloning using LR Clonase II^{66 (link)}. Recombinant adenoviruses were generated in HEK293 cells and purified by cesium chloride gradient. All sequences were verified by the Iowa Institute of Human Genetics, University of Iowa.

Free full text: Click here

Boyer C.K., Bauchle C.J., Zhang J., Wang Y, & Stephens S.B. (2023). Synchronized proinsulin trafficking reveals delayed Golgi export accompanies β-cell secretory dysfunction in rodent models of hyperglycemia. Scientific Reports, 13, 5218.

Publication 2023

sfGFP-C1

Adenoviruses Apai C peptide Cdna Cesium chloride Coding sequence Hek293 cells Human Ires Plasmid Proinsulin

Corresponding Organization :

Other organizations : University of Iowa, Fraternal Order of Eagles, University of Michigan–Ann Arbor

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

Gibson assembly and subcloning of the ProCpepRUSH (SA-KDEL-IRES-proCpep-SBP-GFP) construct into pENTR2b-RIP¹²
Separate amplification of SA-KDEL-IRES and SBP PCR fragments from Str-KDEL_SBP-EGFP-GPI
PCR amplification of sfGFP from sfGFP-C1
PCR amplification of proinsulin as two fragments on either side of the ApaI site from human INS cDNA for insertion of the SBP and sfGFP fragments within the C-peptide coding sequence
Recombination of RIP-SA-KDEL-IRES-proCpep-SBP-GFP from pENTR2b-RIP into a modified pAD-PL/DEST via Gateway cloning using LR Clonase II
Generation of recombinant adenoviruses in HEK293 cells and purification by cesium chloride gradient

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!