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A novel, long-acting agonist of glucose-dependent insulinotropic polypeptide suitable for once-daily administration in type 2 diabetes

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  • Glucose-Dependent Insulinotropic Polypeptide (Gip) Is A Gastrointestinal Hormone With A Potentially
  • Rapid Degradation By Dipeptidylpeptidase Iv Has Prompted The Development Of Enzyme-Resistant N-Termi
  • But Renal Clearance Still Limits In Vivo Bioactivity
  • In This Study
  • We Report Long-Term Antidiabetic Effects Of A Novel
  • N-Terminally Protected
  • Fatty Acid-Derivatized Analog Of Gip
  • N-Acgip(Lyspal(37))
  • In Obese Diabetic (Ob/Ob) Mice
  • Once-Daily Injections Of N-Acgip(Lyspal(37)) Over A 14-Day Period Significantly Decreased Plasma Glu
  • Glycated Hemoglobin
  • And Improved Glucose Tolerance Compared With Ob/Ob Mice Treated With Saline Or Native Gip
  • Plasma Insulin And Pancreatic Insulin Content Were Significantly Increased By N-Acgip(Lyspal(37))
  • This Was Accompanied By A Significant Enhancement In The Insulin Response To Glucose Together With A
  • No Evidence Was Found For Gip Receptor Desensitization And The Metabolic Effects Of Nacgip(Lyspal(37
  • Similar Daily Injections Of Native Gip Did Not Affect Any Of The Parameters Measured
  • These Data Demonstrate The Ability Of Once-Daily Injections Of N-Terminally Modified
  • Fatty Acid-Derivatized Analogs Of Gip
  • Such As N-Acgip(Lyspal(37))
  • To Improve Diabetes Control And To Offer A New Class Of Agents For The Treatment Of Type 2 Diabetes
  • Biology
  • Design
  • Engineering


31 From: Methods in Molecular Biology, vol. 435: Chromosomal Mutagenesis Edited by: G. Davis and K. J. Kayser © Humana Press Inc., Totowa, NJ 3 Robust Cell Line Development Using Meganucleases Jean-Pierre Cabaniols and Frédéric Pâques Summary Cell line development for protein production or for the screening of drug targets requires the reproducible and stable expression of transgenes. Such cell lines can be engineered with meganu- cleases, sequence-specific endonucleases that recognize large DNA target sites. These proteins are powerful tools for genome engineering because they can increase homologous gene targeting by several orders of magnitude in the vicinity of their cleavage site. Here, we describe in details the use of meganucleases for gene targeting in Chinese hamster ovary-K1 cells, with a special emphasis on a gene insertion procedure using a promoter-less marker gene for selection. We have also monitored the expression of genes inserted by meganucleases-induced recombination, and show that expres- sion is reproducible among different targeted clones, and stable over a 4 mo period. These experi- ments were conducted with the natural yeast I-SceI meganuclease, but the general design and process can also be applied to engineered meganucleases. Key Words: Cell line development; double-strand break; gene targeting; homologous recombi- nation; I-SceI; meganucleases; protein production. 1. Introduction Homologous recombination is a powerful tool for genome engineering. Since the first gene targeting experiments in yeast more than 25 yr ago (1,2), homologous recombination (HR) has been used to insert, replace, or delete genomic sequences in a variety of cells (3–5). However, targeted events occur at a very low frequency in mammalian cells. The frequency of HR can be significantly increased by a specific DNA double-strand break (DSB) in the targeted locus (6,7). Such DSBs can be delivered with meganucleases, sequence-specific endonucleases that recog- nize large DNA target si

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