Rapid and efficient enhancer cloning and in vivo screening using the developing chick embryo

Ruth M Williams; Tatjana Sauka-Spengler

doi:10.1016/j.xpro.2021.100507

Rapid and efficient enhancer cloning and in vivo screening using the developing chick embryo

STAR Protoc. 2021 May 5;2(2):100507. doi: 10.1016/j.xpro.2021.100507. eCollection 2021 Jun 18.

Authors

Ruth M Williams¹, Tatjana Sauka-Spengler¹

Affiliation

¹ University of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford OX3 9DS, UK.

Abstract

Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity. For complete details on the use and execution of this protocol, please see Williams et al. (2019).

Keywords: CRISPR; Gene Expression; Model Organisms; Molecular Biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Chick Embryo
Cloning, Molecular*
Enhancer Elements, Genetic*

Grants and funding

G0902418/MRC_/Medical Research Council/United Kingdom