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Cell Penetrating Peptides-CPPs

Cell Penetrating Peptides-CPPs. Petra Oražem , University of Ljubljana, Biotechnical faculty, Slovenia . Introduction. Class of short peptides (10-30 amino acids) with a property to translocate across cell membranes, introduced in the late 1980s

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Cell Penetrating Peptides-CPPs

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  1. Cell Penetrating Peptides-CPPs Petra Oražem, University of Ljubljana, Biotechnical faculty, Slovenia

  2. Introduction • Class of short peptides (10-30 amino acids) with a property to translocate across cell membranes, introduced in the late 1980s • Most of investigations pertaining to CPPs, such as structural and functional studies, have been carried out in the animal systems • CPP uptake pattern between the mammalian system and the plant system was later found to be similar

  3. Subgroups of CPPs • Protein derived (P) • Chimeric (C) • Designed/synthetic (S)

  4. Protein derived CPPs • penetratin-first presented CPP • Tat and Tat2 • pVEC • CPPs can deliver proteins, DNAs and RNAs into cells • The mechanism of uptake of CPP-cargo complexes across the plasma membrane remains unresolved • CPPs have been shown to efficiently improve intracellular delivery of various biomolecules

  5. CPPs and plant systems • Unexplored until the emergence of recent reports focusing on translocation of arginine rich CPPs in plant cells • Dr. François Eudes and his team from Lethbridge Research Centre in Canada, reported of successful protocol for translocation of fluorescently labeled CPPs and delivery of GUS enzyme and linear DNA in triticale microspores (Chugh et al. 2009, Chugh et al. 2010) • They also investigate cellular internalization of pVEC, transportan, penetratin, monomer and dimer of HIV-1 Tat basic domain in tobacco and triticale protoplasts

  6. Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microsporesArchana Chugh, Eric Amundsen and François Eudes, 2009 • Uptake of fluorescently labeled CPPs (Tat, Tat2, M-Tat, pVEC, transportan) in the isolated triticale microspores • Demonstrated that Tat2 is able to efficiently transduce GUS enzyme in its functional form into microspores • ...and show that Tat2 can successfully deliver GUS gene in triticale microspores

  7. Other studies... • Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos (Archana Chugh and François Eudes, 2008) • Cellular uptake of cell-penetrating peptides pVEC and transportan in plants (Archana Chugh and François Eudes, 2008) • Translocation and nuclear accumulation of monomer and dimer of HIV-1 Tat basic domain in triticale mesophyll protoplasts(Archana Chugh and François Eudes, 2007) • Critical Review: Cell penetrating Peptides: nanocarrier for macromolecule delivery in living cells (Archana Chugh, François Eudes, Youn-Seb Shim, 2010) • A novel method of transgene delivery into Triticale plants using the Agrobacterium transferred DNA- derived nano-complex(Alicja Ziemienowicz, Youn-Seb Shim, Aki Matsuoka, François Eudes, Igor Kovalchuk, 2012)

  8. Our work with CPPs • Microspores two way of development: - pollen maturation or - haploid embryogenesis • microspores are an interesting and useful system for developmental and genetic studies • In tobacco, both systems are well established • In our study with CPPs we attempted to use this technology further in Sambucus nigra, in which we developed a reproducible pollen maturation protocol • The aim of our research was transformation of tobacco microspores with Tat2 and plasmid-DNA complex (GUS expression) • We used GUS plasmid SF-16, with dc3 promotor, which was presented as strong pollen-expressed promoter in other studies of microspore transformation by Touraev et al. 1995

  9. Three major steps... • Microspores isolation • CPP + cargo complex formation • Microspore transformation

  10. Microspores isolation • Protocol from Touraev et al. 1999 • Freshly isolated microspores of tobacco in mid-late uninuculate stage • Microspores density was adjusted to 10⁵ cells/ml

  11. CPP + cargo complex formation • Tat2 was used as CPP • Tat2 and cargo (SF-16 dc3 GUS plasmid) were prepared separately and then combined together in a ratio 4:1 • 4 µg of Tat2 peptide in 100 µl H2O (CPP) • 1 µg of GUS dc3 plasmid in 100 µl H2O (cargo) • incubation at room temperature for 0-15 min...

  12. Microspore transformation • Freshly isolated tobacco microspores in determined concentration • Added complex to pelleted microspores and mixed gently • Incubated at room temperature for 15 minutes

  13. Detection of transformed microspores • GUS histochemical assay (staining with X-Gluc) • Observation with light microscope

  14. Conclusions • We demonstrate that Tat2 is able to effiecently transduce SF-16 dc3 plasmid (GUS) in its functional form in the tobacco microspores • Transient transformation was observed (blue color of microspores) • Problems with reproducibility • Reasons? • Several problems during Tat2 and cargo synthesis size of the complex (time?) is crucial for successful cell transformation • Purchasing CPPs from two different manufacturers (problems with delivery?)

  15. Special thanks to... • Dr. François Eudes and his team for support during my work in their laboratory

  16. THANK YOU

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