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Published on: June 14, 2018
Small Interfering RNA Delivery Into Primary Human Natural Killer Cells for Functional Gene Analyses
Pouria Momayyezi1, Karl-Johan Malmberg1,2, Quirin Hammer1
1Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
Abstract:
Studying gene functions in human natural killer (NK) cells is key for advancing the understanding of NK cell biology and holds promise to pave the way for improving NK cell therapies against cancer. However, NK cells are challenging to manipulate, and investigation of gene functions in NK cells is hampered by variable delivery efficiencies and impaired viability upon electroporation, lipofection, or viral transduction. Here, we report a simple workflow for delivery of commercially available small interfering RNA molecules into primary human NK cells to enable functional gene analyses. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Enrichment of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Preparation of small interfering RNA Basic Protocol 3: Delivery of small interfering RNA into natural killer cells Support Protocol 1: Isolation of human peripheral blood mononuclear cells from buffy coats Support Protocol 2: Thawing and recovery of cryopreserved peripheral blood mononuclear cells Support Protocol 3: Evaluation of natural killer cell purity following magnetic enrichment Support Protocol 4: Exemplary assessment of knockdown efficiency.
Insights
This study presents a straightforward method for delivering small interfering RNA (siRNA) into human natural killer (NK) cells. This technique enhances gene function analysis in NK cells, crucial for cancer immunotherapy research.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Understanding gene functions in human natural killer (NK) cells is vital for advancing NK cell biology and cancer immunotherapy.
- Current methods for gene manipulation in NK cells face challenges with delivery efficiency and cell viability.
Purpose of the Study:
- To develop a simple and effective workflow for delivering small interfering RNA (siRNA) into primary human NK cells.
- To facilitate functional gene analyses in NK cells for improved understanding and therapeutic applications.
Main Methods:
- Enrichment of NK cells from peripheral blood mononuclear cells (PBMCs).
- Preparation of siRNA molecules.
- Delivery of siRNA into primary human NK cells using a novel workflow.
- Protocols for cell isolation, thawing, purity assessment, and knockdown efficiency evaluation are detailed.
Main Results:
- A reproducible workflow for delivering siRNA into primary human NK cells was established.
- The method enables effective gene silencing for functional studies in NK cells.
- Improved viability and delivery efficiency compared to traditional methods.
Conclusions:
- The developed workflow simplifies gene function analysis in human NK cells.
- This advancement supports the development of improved NK cell-based cancer therapies.
- The protocol provides a valuable tool for researchers in immunology and cell therapy.
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