دورية أكاديمية
Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.
| العنوان: | Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain. |
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| المؤلفون: | Linesch PW; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California., Akhtar AA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California., Breunig JJ; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California.; Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California.; Division of Applied Cell Biology and Physiology, Cedars-Sinai Medical Center, Los Angeles, California.; Department of Medicine, UCLA, Los Angeles, California. |
| المصدر: | Current protocols [Curr Protoc] 2023 Jun; Vol. 3 (6), pp. e792. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: John Wiley & Sons Country of Publication: United States NLM ID: 101773894 Publication Model: Print Cited Medium: Internet ISSN: 2691-1299 (Electronic) Linking ISSN: 26911299 NLM ISO Abbreviation: Curr Protoc Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Hoboken, NJ : John Wiley & Sons, [2021]- |
| مواضيع طبية MeSH: | Doxycycline*/pharmacology , Doxycycline*/metabolism , Induced Pluripotent Stem Cells*/metabolism, Humans ; Animals ; Mice ; Genes, Reporter ; Genetic Vectors ; DNA Transposable Elements ; Anti-Bacterial Agents/metabolism ; Tetracycline/pharmacology ; Tetracycline/metabolism ; Luciferases/genetics ; Luciferases/metabolism ; Gene Expression ; Brain ; Mammals/genetics ; Mammals/metabolism |
| مستخلص: | Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity. (© 2023 Wiley Periodicals LLC.) |
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| معلومات مُعتمدة: | R33 CA202900 United States CA NCI NIH HHS; R03 NS101529 United States NS NINDS NIH HHS; R33 CA236687 United States CA NCI NIH HHS; R01 NS121617 United States NS NINDS NIH HHS; P50 CA211015 United States CA NCI NIH HHS |
| فهرسة مساهمة: | Keywords: directed differentiation; iPSC; safe harbor site; transgenesis; transposon |
| المشرفين على المادة: | N12000U13O (Doxycycline) 0 (DNA Transposable Elements) 0 (Anti-Bacterial Agents) F8VB5M810T (Tetracycline) EC 1.13.12.- (Luciferases) |
| تواريخ الأحداث: | Date Created: 20230607 Date Completed: 20230609 Latest Revision: 20240923 |
| رمز التحديث: | 20240923 |
| مُعرف محوري في PubMed: | PMC10264152 |
| DOI: | 10.1002/cpz1.792 |
| PMID: | 37283517 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 2691-1299 |
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| DOI: | 10.1002/cpz1.792 |