Dataset Information

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

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).

SUBMITTER: Williams RM

PROVIDER: S-EPMC8121703 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid and efficient enhancer cloning and <i>in vivo</i> screening using the developing chick embryo.

Williams Ruth M RM Sauka-Spengler Tatjana T

STAR protocols 20210505 2

Here, we describe a highly efficient, medium-throughput strategy for cloning and <i>in vivo</i> 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 t ...[more]

PMID: 34027475

Similar Datasets

Project description:Synovial joints are among the most important structures that give us complex motor abilities as humans. Degenerative joint diseases, such as arthritis, cause loss of normal joint functioning and affect over 40 million people in the USA and approximately 350 million people worldwide. Therapies based on regenerative medicine hold the promise of effectively repairing or replacing damaged joints permanently. Here, for the first time, we introduce a model for synovial joint regeneration utilizing the chick embryo. In this model, a block of tissue that contains the prospective elbow is excised, leaving a window with strips of anterior and posterior tissue intact (window excision, WE). In contrast, we also slice out the same area containing the elbow and the distal piece of the limb is pinned back onto the stump (slice excision, SE). Interestingly, when the elbow is removed via WE, regeneration of the joint takes place, whereas the elbow joint does not regenerate following SE. In order to investigate whether the regeneration response recapitulates the developmental program of forming joints, we used GDF-5 and Autotaxin (Atx) as joint tissue specific markers, and Sox-9 and Col-9 as cartilage markers for in situ hybridization on sections at different time points after WE and SE surgeries. Re-expression of GDF-5 and Atx is observed in the WE samples by 60h after surgery. In contrast, the majority of the samples that underwent SE surgery did not express GDF-5 and Atx. Also, in SE fusion of cartilage elements takes place and the joint interzone does not form. This is indicated by continuous Col-9 expression in SE limbs, whereas Col-9 is downregulated at the joint interzone in the regenerating WE samples. This order and pattern of gene expression observed in regenerates is similar to the development of a joint suggesting that regeneration recapitulates development at the molecular level. This model defines some of the conditions required for inducing joint regeneration in an otherwise nonregenerating environment. This knowledge can be useful for designing new therapeutic approaches for joint loss or for conditions affecting joint integrity in humans.

Dataset Information

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

Publications

Rapid and efficient enhancer cloning and <i>in vivo</i> screening using the developing chick embryo.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets