Ap C ntr l or The easy way to success Selection Eukaryotic - PowerPoint PPT Presentation

Ap C ntr l or The easy way to success

Selection Eukaryotic Bacteria cells selection Antibiotic We show you the future of eukaryotic cell transfections …

Our motivation ? Months of work … Is there no other possibility? Ah!...what an idea!

Our Idea Apoptosis as selection marker Transient Stable transfected transfected

What is Apoptosis? Programmed cell death Cell, nuclear and DNA Phagocytosis Normal cell fragmentation Blebbing Figure 21-33 Molecular Cell Biology, Sixth Edition 2008 W.H. Freeman Company

Why apoptosis as a selection marker? Apoptosis Antibiotics Functional principle Programmed cell death Cytotoxicity Effects on neighboring Minimal due to the Very possible due to cells early loss of cell cell lysis connection Duration 1-2 days >2 weeks Satellite colonies Not possible Possible Costs Low High Inducible conditions Wide range of Not possible possibilities

ApoControl: Three systems • Cut‘N‘Survive (transient) • Jump-or-Die (everlasting) • ProSearch

Cut‘N‘Survive • Cut‘N‘Survive (transient) • Cell line with inducible Bak-fusion protein • Plasmid encoding protein of interest and ANTI-Bak-system If the plasmid is transfected and expressed, the cell will be prevented from apoptosis due to the degradation of Bak.

Cut‘N‘Survive Overview • Cut‘N‘Survive (transient) • Untransfected cell: Dies because of Bak-fusion protein • Transfected Cell: Survives because of ANTI-Bak components expressed from the plasmid

Cut‘N‘Survive Without Plasmid C ell WITHOUT plasmid: Apoptosis From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive Bak-protein • Bak • Mitochondrial membrane protein • Key protein to induce apoptosis in mammalian cells

Cut‘N‘Survive Without Plasmid Cell WITHOUT plasmid : Apoptosis From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component + protein of interest From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component + protein of interest Interaction From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component + protein of interest From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive TEV protease Recognition site • TEV protease … … Glu-Asn-Leu-Tyr-Phe-Gln-Gly • Cysteine Protease • Cleavage at a specific sequence

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component + protein of interest From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive Degradation Signal • N-degron • Degradation signal • N-terminal amino acid sequence

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component + protein of interest Interaction From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component + protein of interest TEV Protease cuts recognition sites From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12 Protein of interest TEV Protease cuts recognition sites From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12 Protein of interest TEV Protease cuts recognition sites From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : From the plasmid: BB12, the ANTI-Bak component Protein of interest Degradation because of free N-terminus of N-degron From the genome: BB9, the Bak-fusion protein

Cut‘N‘Survive With Plasmid Cell WITH plasmid : Protein of interest Cell survives with your protein of interest

BioBricks BB9 integrated into the genome … … TEV recognition site BBa_K368009 BB12 in pSB1C3: BBa_K368012

BioBricks BB9 integrated into the genome … … BBa_K368016 BB12 in pSB1C3: BBa_K368011 BBa_K368019

ApoControl: Three systems • Cut‘N‘Survive (transient) • Jump-or-Die (everlasting) • ProSearch

ApoControl: Three systems • Jump-or-Die (everlasting) • Cell line with an inducible Bak-gene • Plasmid with an integrase • Plasmid with your gene of interest If the plasmid with your gene of interest is integrated into the genome, the cell will be prevented from apoptosis by stopping the transcription before the Bak-gene.

Jump-or-Die BB3 integrated into genome … … Cell WITHOUT plasmids: Apoptosis

Jump-or-Die With Plasmid BB3 integrated into genome … … BBa_K368003 BBs 4&6 in pSB1C3: Cell WITH plasmids: BBa_K368006 BBa_K368004

Jump-or-Die With Plasmids Cell WITH plasmids: … … BBa_K368003 BBa_K368004 Integrase

Jump-or-Die With Plasmids Cell WITH plasmids: … … bacterial / phage attachment sites BBa_K368004

Jump-or-Die With Plasmids Cell WITH plasmids: … …

Jump-or-Die With Plasmids Cell WITH plasmids: … …

Jump-or-Die With Plasmids Plasmid integrated in genome: STOP

Jump-or-Die With Plasmids Plasmid integrated in genome: STOP

BioBricks BBa_K368009 BBa_K368006 BBa_K368004 SV40PA

ApoControl: Three systems • Cut‘N‘Survive (transient) • Jump-or-Die (everlasting) • ProSearch

Problem? • What, if your lab doesn‘t support tet-on?

ProSearch • On the database of HEK 293T • Tool to find combinations of inducible promoters, their transkription factors and genes that are naturally expressed • Helps you find your own inducible promoter for special conditions

Perspective • Finish the Biobricks, test them and the whole systems for working and efficiency • Establishing our systems as new selection standards • Model TF activity per environmental condition to find the most important TFs that trigger the corresponding response

We wouldn‘t have come so far without … • Our supervisors: - Prof. Dr. Kirsten Jung - Prof. Dr. Thorsten Mascher - Prof. Dr. Angelika Böttger - Dr. Susanne Gebhard - Dr. Achim Tresch - Kemal Akman • The labs that supported our research - Knop Group, EMBL - Basler Lab, University of Zurich

Thanks to our sponsors!

Sources • Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009) • Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007) • Brown et al (2010): Gene Expression and Transcription Factor Profiling Reveal Inhibition of Transcription Factor cAMP- response Element-binding Protein by {gamma}-Herpesvirus Replication and Transcription Activator. J Biol Chem 2010 Aug 13;285(33):25139-53. • Calzado et al (2009): An inducible autoregulatory loop between HIPK2 and Siah2 at the apex of the hypoxic response. Nat Cell Biol 2009 Jan;11(1):85-91. • Campeau et al (2009): Characterization of Gaucher disease bone marrow mesenchymal stromal cells reveals an altered inflammatory secretome. Blood 2009 Oct 8;114(15):3181-90. • Chamorro et al (2005): FGF-20 and DKK1 are transcriptional targets of beta-catenin and FGF-20 is implicated in cancer and development. EMBO J 2005 Jan 12;24(1):73-84. • Ciuffi et al (2005): A role for LEDGF/p75 in targeting HIV DNA integration. Nat Med 2005 Dec;11(12):1287-9. • Gat-viks et al (2005): The Factor Graph Network Model for Biological Systems, Proc. of RECOMB 2005, doi 10.1.1.109.5329 • Guo et al (2008): Gene transfer: the challenge of regulated gene expression, Trends in Molecular Medicine Volume 14, Issue 9, September 2008, 410-418 • Mering et al. (2003): STRING: a database of predicted functional associations between proteins., Nucleic Acids Res . 2003 Jan 1;31(1):258-61 • Taylor et al (2009): Genome wide analysis of human genes transcriptionally and post-transcriptionally regulated by the HTLV- I protein p30. BMC Genomics 2009 Jul 14;10:311. • Zhao et al (2005): TRED: a Transcriptional Regulatory Element Database and a platform for in silico gene regulation studies, Nucl. Acids Res. 2005 Vol. 33: D103 – D107 • www.pdb.de • http://www.pnas.orgcontent1042711209F1.large.jpg

Feel free to ask questions!