An optimized retroviral toolbox for overexpression and genetic perturbation of primary lymphocytes

Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of genetic modification of primary lymphocytes. Here,...

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Bibliographic Details
Published in:	Biology open Vol. 11; no. 2
Main Authors:	van der Donk, Lieve E H, van der Spek, Jet, van Duivenvoorde, Tom, Ten Brink, Marieke S, Geijtenbeek, Teunis B H, Kuijl, Coenraad P, van Heijst, Jeroen W J, Ates, Louis S
Format:	Journal Article
Language:	English
Published:	England The Company of Biologists Ltd 15-02-2022 The Company of Biologists
Subjects:	Animals Antibiotic resistance Antibiotics Antigens Construction Expression vectors Fluorescence Genetic modification Genetic Vectors - genetics Humans Lymphocytes Lymphocytes T Methods & Techniques Mice Optimization overexpression Perturbation Promoter Regions, Genetic Promoters Retroviridae - genetics retrovirus Streamlining Surface markers t cell Retrovirus Overexpression Lymphocytes T cell
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Summary:	Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of genetic modification of primary lymphocytes. Here, we report the construction and validation of a versatile range of retroviral expression vectors. These vectors can be used for the knockdown or overexpression of genes of interest in primary human and murine lymphocytes, in combination with a wide choice of selection and reporter strategies. By streamlining the vector backbone and insert design, these publicly available vectors allow easy interchangeability of the independent building blocks, such as different promoters, fluorescent proteins, surface markers and antibiotic resistance cassettes. We validated these vectors and tested the optimal promoters for in vitro and in vivo overexpression and knockdown of the murine T cell antigen receptor. By publicly sharing these vectors and the data on their optimization, we aim to facilitate genetic modification of primary lymphocytes for researchers entering this field.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Present address: Neogene Therapeutics, Science Park 106, 1098 XG, Amsterdam, the Netherlands.
ISSN:	2046-6390 2046-6390
DOI:	10.1242/bio.059032