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Summer Research Fellowship Programme of India's Science Academies

Generation of Rab7L1 Knock-Down Stable THP-1 Cell Lines to Study Its Role in Host Immune Response

Achyut Bora

School Of Biotechnology, Banaras Hindu University, Varanasi

Dr Sangita Mukhopadhyay

Staff Scientist – VI and Group Leader, Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting & Diagnostics, (CDFD) Hyderabad

ABSTRACT

RAB7L1 is known as Rab7 like protein 1 and is encoded by the RAB7L1 gene. RAB7L1, a small GTP binding protein, is a member of the RAS oncogene superfamily. Rab7L1 protein has been recently demonstrated to regulate phagocytosis and cellular traffic from Golgi to lysosome.  Earlier studies from our lab have shown that Rab7L1 gets recruited to phagosome and plays a critical role in regulating phagosome-lysosome fusion (P-L fusion) process. Inspite of extensive research, the molecular mechanisms involving Rab7L1 and P-L fusion have not been completely elucidated. Phagosome-lysosome fusion plays a central role in innate defense mechanisms and regulates various cellular functions like autophagy, antigen presentation, and apoptosis. To determine the role of Rab7L1 in the regulation of host immune functions, our primary objective is to establish a Rab7L1 knock-down stable THP-1 cell line.  Rab7L1-specific shRNA cloned in the pLKO.1 vector was co-transfected along with VSV-G and VSV-P packaging plasmid. Lentivirus packaged with Rab7L1 specific shRNA was used to prepare Rab7L1-KD THP-1 monocyte by selecting them using puromycin.  To confirm knockdown expression of Rab7L1, Rab7L1-KD cells will be compared with scrambled THP-1 cells at mRNA level by RT-PCR and at the protein level by immunoblotting using anti-Rab7L1 Ab. Rab7L1-KD THP-1 macrophages will be further used to study various aspect of immunological functions viz  effect of Rab7L1-KD on the survival of THP-1 macrophages by MTT assay, cytokine profiling by ELISA and role of Rab7L1 in PMA mediated THP-1 macrophage differentiation. This will help us to come to a conclusion on the involvement of Rab7L1 in these cellular process.   

Keywords: Phagosome-Lysosome fusion, RAB7L1 (Ras-related protein Rab-7 like 1 protein), THP-1 (Human monocytic cell line), short hairpin RNA (shRNA), RT-PCR, PMA (Phorbol myristate acetate)

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