A rapid and robust protocol for generating loss-of-function alleles in pluripotent stem cells
Summary: The generation of loss-of-function alleles in human pluripotent stem cells (hPSCs) is used to interrogate gene function and validate reagents; however, identifying clones harboring true loss-of-function alleles remains inefficient. To address this, we present BOLT (barcoded oligos for loss-...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725003223 |
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| Summary: | Summary: The generation of loss-of-function alleles in human pluripotent stem cells (hPSCs) is used to interrogate gene function and validate reagents; however, identifying clones harboring true loss-of-function alleles remains inefficient. To address this, we present BOLT (barcoded oligos for loss-of-function targeting), a streamlined protocol that simplifies the screening process, facilitating rapid validation of loss-of-function mutations. We describe steps for designing editing tools, nucleofection, and clonal density plating. We then detail procedures for bridging PCR, isolating clones derived from a single hPSC, single-clone screening, and Sanger barcode detection.For complete details on the use and execution of this protocol, please refer to Matera et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
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| ISSN: | 2666-1667 |