Abstract
We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants. Using the experimental procedures and conditions described here, as high as 20% of bombarded embryos can produce regenerable recombinant callus events. This nanomaterial-mediated, DNA-free methodology has potential to become an effective tool for plant genome editing.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Wang Y, Yau Y-Y, Perkins-Balding D, Thomson J (2011) Recombinase technology: applications and possibilities. Plant Cell Rep 30:267–285
Baubonis W, Sauer B (1993) Genomic targeting with purified Cre recombinase. Nucleic Acids Res 21:2025–2029
Luckow B, Hänggli A, Maier H, Chilla S, Loewe RP, Dehmel S, Schlöndorff D, Loetscher P, Zerwes H-G, Müller M (2009) Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice. Genesis 47:545–558
de Wit T, Drabek D, Grosveld F (1998) Microinjection of Cre recombinase RNA induces site-specific recombination of a transgene in mouse oocytes. Nucleic Acids Res 26:676–678
Kolb AF, Siddell SG (1996) Genomic targeting with an MBP-Cre fusion protein. Gene 183:53–60
Ponsaerts P, Brown JP, Van den Plas D, Van den Eeden L, Van Bockstaele DR, Jorens PG, Van Tendeloo VFI, Merregaert J, Singh PB, Berneman ZN (2004) Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination. Gene Ther 11:1606–1610
Vergunst AC, Schrammeijer B, den Dulk-Ras A, de Vlaam CMT, Regensburg-Tuïnk TJG, Hooykaas PJJ (2000) VirB/D4-dependent protein translocation from agrobacterium into plant cells. Science 290:979–982
Koshy AA, Fouts AE, Lodoen MB, Alkan O, Blau HM, Boothroyd JC (2010) Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions. Nat Methods 7:307–309
Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE (2001) Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. Nat Biotechnol 19:929–933
Will E, Klump H, Heffner N, Schwieger M, Schiedlmeier B, Ostertag W, Baum C, Stocking C (2002) Unmodified Cre recombinase crosses the membrane. Nucleic Acids Res 30:e59
Lin Q, Jo D, Gebre-Amlak K, Ruley HE (2004) Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells. BMC Biotechnol 4:25
Nolden L, Edenhofer F, Haupt S, Koch P, Wunderlich FT, Siemen H, Brustle O (2006) Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase. Nat Methods 3:461–467
Wymer CL, Fernández-Ábalos JM, Doonan JH (2001) Microinjection reveals cell-to-cell movement of green fluorescent protein in cells of maize coleoptiles. Planta 212:692–695
Wu J, Du H, Liao X, Zhao Y, Li L, Yang L (2011) An improved particle bombardment for the generation of transgenic plants by direct immobilization of relleasable Tn5 transposases onto gold particles. Plant Mol Biol 77:117–127
Chugh A, Eudes F, Shim Y-S (2010) Cell-penetrating peptides: nanocarrier for macromolecule delivery in living cells. IUBMB Life 62:183–193
Cao M-X, Huang J-Q, Yao Q-H, Liu S-J, Wang C-L, Wei Z-M (2006) Site-specific DNA excision in transgenic rice with a cell-permeable Cre recombinase. Mol Biotechnol 32:55–63
Kim T-W, Slowing II, Chung P-W, Lin VS-Y (2010) Ordered Mesoporous polymer−silica hybrid nanoparticles as vehicles for the intracellular controlled release of macromolecules. ACS Nano 5:360–366
Torney F, Trewyn BG, Lin VSY, Wang K (2007) Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2:295–300
Martin-Ortigosa S, Valenstein JS, Lin VSY, Trewyn BG, Wang K (2012) Gold functionalized Mesoporous silica nanoparticle mediated protein and DNA Codelivery to plant cells via the biolistic method. Adv Funct Mater 22:3576–3582
Martin-Ortigosa S, Valenstein JS, Sun W, Moeller L, Fang N, Trewyn BG, Lin VSY, Wang K (2012) Parameters affecting the efficient delivery of Mesoporous silica nanoparticle materials and gold Nanorods into plant tissues by the biolistic method. Small 8:413–422
Martin-Ortigosa S, Peterson DJ, Valenstein JS, Lin VS-Y, Trewyn BG, Lyznik LA, Wang K (2014) Mesoporous silica nanoparticle-mediated intracellular Cre protein delivery for maize genome editing via loxP site excision. Plant Physiol 164:537–547
Slowing II, Trewyn BG, Lin VSY (2007) Mesoporous silica nanoparticles for intracellular delivery of membrane-impermeable proteins. J Am Chem Soc 129:8845–8849
Chu CC, Wang CC, Sun CS, Chen H, Yin KC, Chu CY, Bi FY (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on nitrogen-sources. Sci Sinica 18:659–668
Vain P, McMullen MD, Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12:84–88
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Frame BR, Zhang HY, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE, Zhen SF, Schnable PS, Wang K (2000) Production of transgenic maize from bombarded type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev-Pl 36:21–29
Greenhouse care for transgenic maize plants (2009) Department of Agronomy, Plant Transformation Facility, Iowa State University. http://www.agron.iastate.edu/ptf/protocol/Greenhouse%20Protocol.pdf
Abremski K, Hoess R (1984) Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. J Biol Chem 259:1509–1514
Cantor EJ, Chong S (2001) Intein-mediated rapid purification of Cre Recombinase. Protein Expr Purif 22:135–140
Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973
Komari T, Kubo T (1999) Methods of genetic transformation: agrobacterium tumefaciens. In: Vasil I (ed) Molecular improvement of cereal crops, vol 5. Advances in cellular and molecular biology of plants. Springer, Dordrecht, pp 43–82
Acknowledgments
This article is dedicated to the memory of Professor Victor S.-Y. Lin, deceased May 4, 2010, in recognition of his inspiration and initial preparation of this work. This work was partially supported by Iowa State University Plant Sciences Institute and DuPont Pioneer. The authors thank Justin Valenstein, Alexander Lyznik, and David Peterson for their scientific and technical inputs and collaboration.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Martin-Ortigosa, S., Trewyn, B.G., Wang, K. (2017). Nanoparticle-Mediated Recombinase Delivery into Maize. In: Eroshenko, N. (eds) Site-Specific Recombinases. Methods in Molecular Biology, vol 1642. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7169-5_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7169-5_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7167-1
Online ISBN: 978-1-4939-7169-5
eBook Packages: Springer Protocols