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Methods Mol Biol. 2016;1407:169-77. doi: 10.1007/978-1-4939-3480-5_13.

shRNA-Induced Gene Knockdown In Vivo to Investigate Neutrophil Function.

Author information

1
Department of Pharmacology, Vascular Biology and Therapeutic Program, Yale University School of Medicine, 10 Amistad, P.O. Box 208089, New Haven, CT, 06520, USA.
2
Department of Pharmacology, Vascular Biology and Therapeutic Program, Yale University School of Medicine, 10 Amistad, P.O. Box 208089, New Haven, CT, 06520, USA. dianqing.wu@yale.edu.

Abstract

To silence genes in neutrophils efficiently, we exploited the RNA interference and developed an shRNA-based gene knockdown technique. This method involves transfection of mouse bone marrow-derived hematopoietic stem cells with retroviral vector carrying shRNA directed at a specific gene. Transfected stem cells are then transplanted into irradiated wild-type mice. After engraftment of stem cells, the transplanted mice have two sets of circulating neutrophils. One set has a gene of interest knocked down while the other set has full complement of expressed genes. This efficient technique provides a unique way to directly compare the response of neutrophils with a knocked-down gene to that of neutrophils with the full complement of expressed genes in the same environment.

KEYWORDS:

Gene knockdown; Neutrophils; RNA interference; Retroviral vectors; Short hairpin RNA; Transplantation

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