Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session II
36 – POS
Board 8
Integrin Alpha-4 Subunit Gene Expression in Mesenchymal Stem Cells using Clinically
Applicable mRNA-based Approach
Adam Nowakowski
1
, Anna Andrzejewska
1
, Piotr Walczak
2
, Barbara Lukomska
1
, Miroslaw
Janowski
2,1
.
1
MMRC PAS, Warsaw, Poland,
2
The Johns Hopkins University School of Medicine, Baltimore,
MD, USA.
Mesenchymal stem cells (MSC) transplantation was found as a new approach to repair tissues
and blood-based delivery is suggested as a minimal invasive approach. α4β1 integrin is involved
in leukocyte extravasation. It is plausible to verify whether the increase of such molecules in the
cell membrane could enable migration of exogenous engineered cells into the injured tissue. The
aim of our work was to elicit overexpression of the ITGA4gene in MSC by mRNA mediated
transfection.
ITGA4gene cDNA was cloned to pSP72vector (P2191-Promega) and used as a template for
mRNA production in vitro. T7mMessage-mMachine Kit (AM1344,-Ambion) with poly(A)
tailing kit (AM1350-Ambion) and mMessage mMachine®T7UltraKit (AM1345-Ambion)
including ARCA cap were employed. SSBprotein (S3917-Sigma) was used for mRNA
stabilization. HumanMSC (PT2501-Lonza) and HEK293cells were transfected with
Lipofectamine®2000(Invitrogen). Transfection efficacy was assessed by RT-PCR and ITGA4
protein production was confirmed by ICC.
mRNA-ITGA4 was produced in vitro by T7mMessage-mMachineKit, but no ITGA4protein
production was detected although mRNA-ITGA4 cellular delivery. mRNA-ITGA4 stabilization
by SSBprotein resulted in ITGA4protein synthesis in HEK293cells only. The use of
(ARCA)mRNA-ITGA4 containing anti-reverse-cap-analog(ARCA) resulted in ITGA4protein
detection in MSC. The ITGA4protein MSC distribution was transient and gradually moving
from inner cellular structures toward membrane where was present for up to 24h.
Cytoplasmic mRNA half-time and translation rate pose problems in mRNA-based transfections.
The employment of ARCAcap seems to address some of these questions. Future studies will be
performed to clarify whether this transient ITGA4protein presence in transfected cells is
sufficient for the improvement of their migration from blood to injured tissue.
Supported by a National Centre for Research and Development grant No101 in ERA-NET
NEURON project: "MEMS-IRBI".
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