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Methods Mol Biol. 2019;1944:3-15. doi: 10.1007/978-1-4939-9095-5_1.

Cloning and Mutagenesis Strategies for Large Collagens.

Author information

1
Department of Dermatology, Faculty of Medicine, Medical Center, University of Freiburg, Freiburg, Germany.
2
Department of Dermatology, Faculty of Medicine, Medical Center, University of Freiburg, Freiburg, Germany. alexander.nystroem@uniklinik-freiburg.de.

Abstract

The size and relatively high GC content of cDNAs are challenges for efficient targeted engineering of large collagens. There are both basic biological and therapeutic interests in the ability to modify collagens, as this would allow for studies precisely describing interactions of collagens with specific interaction partners, addressing consequences of individual disease-causing mutations, and assessing therapeutic applicability of precision medicine approaches. Using collagen VII as an example, we will here describe a strategy for rapid and simple modification of cDNAs encoding large collagens. The method is flexible and can be used for the creation of point mutations, small or large deletions, and insertion of DNA.

KEYWORDS:

Collagen VII; Collagenopathy; Dystrophic epidermolysis bullosa; Exon skipping; Gibson assembly

PMID:
30840231
DOI:
10.1007/978-1-4939-9095-5_1
[Indexed for MEDLINE]

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