Making piRNAs in vitro

Kawaoka, S., Katsuma, S., Tomari, Y. (2014) Making piRNAs in vitro. Methods in Molecular Biology, 1093. pp. 35-46. ISSN 10643745 (ISSN); 9781627036931 (ISBN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/24178555
DOI: 10.1007/978-1-62703-694-8_4

Abstract

Classical biochemical approaches have made great contribution to our current understanding of how small interfering RNA (siRNA) and microRNA (miRNA) are produced and function. However, it has been challenging to establish in vitro systems that can dissect the mechanism of PIWI-interacting RNAs (piRNAs), largely due to the lack of suitable cultured cell lines possessing the piRNA pathway endogenously. The silkworm ovary-derived BmN4 cell line is an emerging model for studying piRNAs, because this cell line harbors a fully functional germline piRNA pathway. We recently reported in vitro recapitulation of a part of piRNA biogenesis - loading of precursor piRNAs and 3′-end maturation - by using lysate prepared from BmN4 cells. Here we describe the methods for maintenance of BmN4 cells, preparation of cell lysate, and in vitro assays to dissect piRNA biogenesis. © 2014 Springer Science+Business Media, LLC.

Item Type: Paper
Uncontrolled Keywords: BmN4 cell line Cell culture In vitro system PIWI PIWI-interacting RNAs Silkworm
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > Piwi protein
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > piRNA
CSHL Authors:
Communities: CSHL labs > Vakoc lab
Depositing User: Matt Covey
Date Deposited: 04 Feb 2014 15:45
Last Modified: 04 Feb 2014 15:45
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29468

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