Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

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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