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Collagen pp 3-15 | Cite as

Cloning and Mutagenesis Strategies for Large Collagens

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1944)

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.

Key words

Collagen VII Gibson assembly Dystrophic epidermolysis bullosa Collagenopathy Exon skipping 
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Notes

Acknowledgments

Part of the work was financed by a research collaboration with ProQR, Netherlands. AN’s research was supported by grants from the German Research Foundation, DFG (grants NY90/2-1, NY90/3-2, SFB850-B11 to AN, BR1475/12-1 to LBT), and from the Dystrophic Epidermolysis Bullosa Research Association (DEBRA) (grant Nystrom Bruckner-Tuderman 1) to AN.

References

  1. 1.
    Phillips CL, Lever LW, Pinnell SR et al (1991) Construction of a full-length murine pro alpha 2(I) collagen cDNA by the polymerase chain reaction. J Invest Dermatol 97:980–984CrossRefGoogle Scholar
  2. 2.
    Bornert O, Kühl T, Bremer J et al (2016) Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy. Mol Ther J Am Soc Gene Ther 24:1302–1311.  https://doi.org/10.1038/mt.2016.92CrossRefGoogle Scholar
  3. 3.
    Chmel N, Bornert O, Hausser I et al (2018) Large deletions targeting the triple-helical domain of collagen VII Lead to mild Acral dominant dystrophic Epidermolysis Bullosa. J Invest Dermatol 138:987–991.  https://doi.org/10.1016/j.jid.2017.11.014CrossRefPubMedGoogle Scholar
  4. 4.
    Nyström A, Bornert O, Kühl T et al (2018) Impaired lymphoid extracellular matrix impedes antibacterial immunity in epidermolysis bullosa. Proc Natl Acad Sci U S A 115:E705–E714.  https://doi.org/10.1073/pnas.1709111115CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chen M, Costa FK, Lindvay CR et al (2002) The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa. J Biol Chem 277:2118–2124.  https://doi.org/10.1074/jbc.M108779200CrossRefPubMedGoogle Scholar
  6. 6.
    Nyström A, Bruckner-Tuderman L, Kern JS (2013) Cell- and protein-based therapy approaches for epidermolysis bullosa. Methods Mol Biol Clifton NJ 961:425–440.  https://doi.org/10.1007/978-1-62703-227-8_29CrossRefGoogle Scholar
  7. 7.
    Fritsch A, Spassov S, Elfert S et al (2009) Dominant-negative effects of COL7A1 mutations can be rescued by controlled overexpression of normal collagen VII. J Biol Chem 284:30248–30256.  https://doi.org/10.1074/jbc.M109.045294CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361.  https://doi.org/10.1016/B978-0-12-385120-8.00015-2CrossRefPubMedGoogle Scholar
  9. 9.
    Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345.  https://doi.org/10.1038/nmeth.1318CrossRefPubMedGoogle Scholar
  10. 10.
    Ricard-Blum S (2011) The collagen family. Cold Spring Harb Perspect Biol 3:a004978.  https://doi.org/10.1101/cshperspect.a004978CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Varki R, Sadowski S, Uitto J, Pfendner E (2007) Epidermolysis bullosa II. Type VII collagen mutations and phenotype-genotype correlations in the dystrophic subtypes. J Med Genet 44:181–192.  https://doi.org/10.1136/jmg.2006.045302CrossRefPubMedGoogle Scholar
  12. 12.
    Chen M, Keene DR, Costa FK et al (2001) The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis Bullosa acquisita autoantibodies. J Biol Chem 276:21649–21655.  https://doi.org/10.1074/jbc.M100180200CrossRefPubMedGoogle Scholar
  13. 13.
    Rattenholl A, Pappano WN, Koch M et al (2002) Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen. J Biol Chem 277:26372–26378.  https://doi.org/10.1074/jbc.M203247200CrossRefPubMedGoogle Scholar
  14. 14.
    Moali C, Font B, Ruggiero F et al (2005) Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1-dependent activity to be enhanced by PCPE-1. J Biol Chem 280:24188–24194.  https://doi.org/10.1074/jbc.M501486200CrossRefPubMedGoogle Scholar
  15. 15.
    Nyström A, Velati D, Mittapalli VR et al (2013) Collagen VII plays a dual role in wound healing. J Clin Invest 123:3498–3509.  https://doi.org/10.1172/JCI68127CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bruckner-Tuderman L (2010) Dystrophic epidermolysis bullosa: pathogenesis and clinical features. Dermatol Clin 28:107–114.  https://doi.org/10.1016/j.det.2009.10.020CrossRefPubMedGoogle Scholar
  17. 17.
    Nyström A, Bernasconi R, Bornert O (2018) Therapies for genetic extracellular matrix diseases of the skin. Matrix Biol 71–72:330–347.  https://doi.org/10.1016/j.matbio.2017.12.010CrossRefPubMedGoogle Scholar
  18. 18.
    McGrath JA, Ashton GH, Mellerio JE et al (1999) Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations. J Invest Dermatol 113:314–321.  https://doi.org/10.1046/j.1523-1747.1999.00709.xCrossRefPubMedGoogle Scholar
  19. 19.
    Schwieger-Briel A, Weibel L, Chmel N et al (2015) A COL7A1 variant leading to in-frame skipping of exon 15 attenuates disease severity in recessive dystrophic epidermolysis bullosa. Br J Dermatol 173:1308–1311.  https://doi.org/10.1111/bjd.13945CrossRefPubMedGoogle Scholar
  20. 20.
    Cserhalmi-Friedman PB, McGrath JA, Mellerio JE et al (1998) Restoration of open reading frame resulting from skipping of an exon with an internal deletion in the COL7A1 gene. Lab Investig J Tech Methods Pathol 78:1483–1492Google Scholar
  21. 21.
    Koga H, Hamada T, Ishii N et al (2011) Exon 87 skipping of the COL7A1 gene in dominant dystrophic epidermolysis bullosa. J Dermatol 38:489–492.  https://doi.org/10.1111/j.1346-8138.2010.01008.xCrossRefPubMedGoogle Scholar
  22. 22.
    Goto M, Sawamura D, Nishie W et al (2006) Targeted skipping of a single exon harboring a premature termination codon mutation: implications and potential for gene correction therapy for selective dystrophic epidermolysis bullosa patients. J Invest Dermatol 126:2614–2620.  https://doi.org/10.1038/sj.jid.5700435CrossRefPubMedGoogle Scholar
  23. 23.
    Wu W, Lu Z, Li F et al (2017) Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model. Proc Natl Acad Sci U S A 114:1660–1665.  https://doi.org/10.1073/pnas.1614775114CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Turczynski S, Titeux M, Tonasso L et al (2016) Targeted exon skipping restores type VII collagen expression and anchoring fibril formation in an in vivo RDEB model. J Invest Dermatol 136:2387–2395.  https://doi.org/10.1016/j.jid.2016.07.029CrossRefPubMedGoogle Scholar
  25. 25.
    Bremer J, Bornert O, Nyström A et al (2016) Antisense oligonucleotide-mediated exon skipping as a systemic therapeutic approach for recessive dystrophic Epidermolysis Bullosa. Mol Ther Nucleic Acids 5:e379.  https://doi.org/10.1038/mtna.2016.87CrossRefPubMedGoogle Scholar
  26. 26.
    Toh ZYC, Thandar Aung-Htut M, Pinniger G et al (2016) Deletion of Dystrophin in-frame exon 5 leads to a severe phenotype: guidance for exon skipping strategies. PLoS One 11:e0145620.  https://doi.org/10.1371/journal.pone.0145620CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kühl T, Mezger M, Hausser I et al (2015) High local concentrations of intradermal MSCs restore skin integrity and facilitate wound healing in dystrophic Epidermolysis Bullosa. Mol Ther J Am Soc Gene Ther 23:1368–1379.  https://doi.org/10.1038/mt.2015.58CrossRefGoogle Scholar
  28. 28.
    Wullink B, Pas HH, Van der Worp RJ et al (2018) Type VII collagen in the human accommodation system: expression in Ciliary body, Zonules, and lens capsule. Invest Ophthalmol Vis Sci 59:1075–1083.  https://doi.org/10.1167/iovs.17-23425CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Dermatology, Faculty of Medicine, Medical CenterUniversity of FreiburgFreiburgGermany

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