TY - JOUR
T1 - An optimized retroviral toolbox for overexpression and genetic perturbation of primary lymphocytes
AU - van der Donk, Lieve E. H.
AU - van der Spek, Jet
AU - van Duivenvoorde, Tom
AU - ten Brink, Marieke S.
AU - Geijtenbeek, Teunis B. H.
AU - Kuijl, Coenraad P.
AU - van Heijst, Jeroen W. J.
AU - Ates, Louis S.
N1 - Funding Information: Open Access funding provided by Amsterdam UMC, University of Amsterdam, location AMC. Deposited in PMC for immediate release. L.E.H.v.d.D., J.v.d.S., J.W.J.v.H. and L.S.A. were supported by NWO-VIDI grant 91717305 to J.W.J.v.H.). L.S.A. was further supported by a postdoctoral stipend from the Amsterdam Infection and Immunity Institute. Publisher Copyright: © 2022. Published by The Company of Biologists Ltd.
PY - 2022/2/1
Y1 - 2022/2/1
N2 - 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.
AB - 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.
KW - Lymphocytes
KW - Overexpression
KW - Retrovirus
KW - T cell
UR - http://www.scopus.com/inward/record.url?scp=85125857592&partnerID=8YFLogxK
U2 - https://doi.org/10.1242/bio.059032
DO - https://doi.org/10.1242/bio.059032
M3 - Article
C2 - 35229875
SN - 2046-6390
VL - 11
JO - Biology open
JF - Biology open
IS - 2
M1 - bio059032
ER -