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

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

Sep 28, 2022 325 likes •789 views

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

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  • r

The easy way to success

Ap C ntr l

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Selection

We show you the future of eukaryotic cell transfections…

Antibiotic Eukaryotic cells Bacteria

selection

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Our motivation

Months of work…

?

Is there no other possibility? Ah!...what an idea!

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Our Idea

Apoptosis as selection marker Stable transfected Transient transfected

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Programmed cell death

What is Apoptosis?

Normal cell Cell, nuclear and DNA fragmentation Blebbing Phagocytosis

Figure 21-33 Molecular Cell Biology, Sixth Edition 2008 W.H. Freeman Company
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Why apoptosis as a selection marker?

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

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  • Cut‘N‘Survive (transient)
  • Jump-or-Die (everlasting)
  • ProSearch

ApoControl: Three systems

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  • Cut‘N‘Survive (transient)

Cut‘N‘Survive

  • 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.

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  • Cut‘N‘Survive (transient)
  • Untransfected cell: Dies because
  • f Bak-fusion protein
  • Transfected Cell: Survives because
  • f ANTI-Bak components

expressed from the plasmid

Cut‘N‘Survive Overview

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Cut‘N‘Survive Without Plasmid

Cell WITHOUT plasmid:

Apoptosis

From the genome: BB9, the Bak-fusion protein

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  • Bak

Cut‘N‘Survive Bak-protein

  • Mitochondrial membrane protein
  • Key protein to induce apoptosis in

mammalian cells

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Cut‘N‘Survive Without Plasmid

Cell WITHOUT plasmid:

Apoptosis

From the genome: BB9, the Bak-fusion protein

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Cut‘N‘Survive With Plasmid

Cell WITH plasmid:

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component + protein of interest

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Cut‘N‘Survive With Plasmid

Interaction

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component + protein of interest

Cell WITH plasmid:

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Cut‘N‘Survive With Plasmid

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component + protein of interest

Cell WITH plasmid:

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  • TEV protease

Cut‘N‘Survive TEV protease

  • Cysteine Protease
  • Cleavage at a specific sequence

Glu-Asn-Leu-Tyr-Phe-Gln-Gly

… …

Recognition site
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Cut‘N‘Survive With Plasmid

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component + protein of interest

Cell WITH plasmid:

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  • N-degron

Cut‘N‘Survive Degradation Signal

  • Degradation signal
  • N-terminal amino acid sequence
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Cut‘N‘Survive With Plasmid

Cell WITH plasmid:

Interaction

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component + protein of interest

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Cut‘N‘Survive With Plasmid

TEV Protease cuts recognition sites

Cell WITH plasmid:

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component + protein of interest

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Cut‘N‘Survive With Plasmid

TEV Protease cuts recognition sites

Cell WITH plasmid:

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12 Protein of interest

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Cut‘N‘Survive With Plasmid

TEV Protease cuts recognition sites

Cell WITH plasmid:

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12 Protein of interest

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Cut‘N‘Survive With Plasmid

Degradation because of free N-terminus of N-degron

Cell WITH plasmid:

From the genome: BB9, the Bak-fusion protein From the plasmid: BB12, the ANTI-Bak component Protein of interest

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Cut‘N‘Survive With Plasmid

Cell survives with your protein of interest

Cell WITH plasmid:

Protein of interest

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BioBricks

BB9 integrated into the genome

… …

BB12 in pSB1C3:

BBa_K368009 BBa_K368012 TEV recognition site

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BioBricks

BB9 integrated into the genome BB12 in pSB1C3:

… …

BBa_K368019 BBa_K368011 BBa_K368016

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  • Cut‘N‘Survive (transient)
  • Jump-or-Die (everlasting)
  • ProSearch

ApoControl: Three systems

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  • Jump-or-Die (everlasting)

ApoControl: Three systems

  • 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.

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Jump-or-Die

BB3 integrated into genome

… …

Cell WITHOUT plasmids:

Apoptosis

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Jump-or-Die With Plasmid

BB3 integrated into genome BBs 4&6 in pSB1C3:

… …

Cell WITH plasmids:

BBa_K368004 BBa_K368006 BBa_K368003

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Jump-or-Die With Plasmids

… …

Integrase Cell WITH plasmids:

BBa_K368004 BBa_K368003

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Jump-or-Die With Plasmids

… …

bacterial / phage attachment sites Cell WITH plasmids:

BBa_K368004

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Jump-or-Die With Plasmids

Cell WITH plasmids:

… …

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Jump-or-Die With Plasmids

… …

Cell WITH plasmids:

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Jump-or-Die With Plasmids

Plasmid integrated in genome:

STOP

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Jump-or-Die With Plasmids

Plasmid integrated in genome:

STOP

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BioBricks

SV40PA

BBa_K368004 BBa_K368009 BBa_K368006

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  • Cut‘N‘Survive (transient)
  • Jump-or-Die (everlasting)
  • ProSearch

ApoControl: Three systems

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

Problem?

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  • 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

ProSearch

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  • 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 Perspective

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  • 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

We wouldn‘t have come so far without …

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Thanks to our sponsors!

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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
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Feel free to ask questions!