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piggyBac Stem Cell Engineering Sublicenses Available
| Date post: | 24-Jun-2015 |
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Technology |
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piggyBac Stem Cell Engineering Sublicenses Available
Individual patient, patient population or existing cell lines
piggyBac-mediated insertion/excision for reprogramming
iPS CellsES Cells
Product for sale
In-house research
piggyBac-mediated insertion/excision for differentiation
Differentiated Primary Cells
Product for sale
In-house research HTS, HCS
Extrapolate pre-clinical data in vivo using Transposagen TKO™ and TGEM™ Rat Models
Regenerative Medicine
-10 -3 0 8-10 20
-1 0 1 7 14
Retrovirus Timeline30 Total Days
piggyBac Timeline15 Total Days
PB-mediated reprogramminginvolves the expression ofreprogramming factors
Reprogrammed iPS cells activate and express pluripotent cell surface markers such as Nanog, SSEA1 and Alkaline phosphatase (AP)
Woltjen et al. (2009) Nature 458, 766-771
Re-excision sequence analysis reveals that no mutations are left behind in the genome of reprogrammed iPS cells
PB Transposase
PB recipient plasmid
Following iPS cell reprogramming, re-excision completely removes the reprogramming factors
Woltjen et al. (2009) Nature 458, 766-771
Pluripotent markers remain in iPS cells where the reprogramming factors have been removed.
Woltjen et al. (2009) Nature 458, 766-771
R1 ES cells and rtTA-MEF cells act as positive and negative controls
2.5 x 105 stem cells were transfected with (A) 1.0 mg piggyBac-neo or(B) 1.0 mg piggyBac-neo plus 1.5 mg piggyBac transposase usingTransfectinTM reagent. After 2 weeks of G418 selection, cultures werefixed to determine stable transfection efficiencies.
Transduction efficiencies approaching 5% can be obtained
Pre-published data Ruiz et al. Transposagen Labs 2010
piggyBac (PB) introduces EGFP and RFP to the genomes of multiple hES cell lines and non-human primate cell lines more efficiently than viral vectors and other transposons
EGFP and RFP being expressed in different human ES cell lines and Macacca lines
-PB-mediated genetic modification of ES cells does not exhibit “silencing” or loss of the transgene. After 25 passages EGFP and RFP is still expressed in 99.9% of all cells.
-PB is capable of delivering up to 18 kb inserts into the ES genome which eclipses viral vector and other transposon “pay-load”
-PB transduction efficiency reaches 5% of all transfected cells and PB transgene expression efficiency reaches 90% when using 3 kb inserts and is still an impressive 72% when using 14 kb inserts. These gene delivery efficiencies are far greater than viral vectors are capable of achieving.
Lacoste et al. (2009) Cell Stem Cell 5, 332-342.
-RFP expressing transgenic ES cells
-RFP expression is removed in hES cells be re-expressing PB transposase. The PB transposon is transferred from the hES genome to a recipient plasmid
-Sequencing of the PB insertion site before and after excision reveals complete removal of transposon without leaving a mutation or “footprint”
Lacoste et al. (2009) Cell Stem Cell 5, 332-342.
-PB introduces a dox-inducible shRNA element targeted against pluripotency markers
-The PB-shRNA system decreases pluripotent marker expression while differentiation marker expression is increased
-Dox-shRNA drives nonspecific differentiation of hES cells as seen in the flattened cell formation which represent epithelial like cells
Lacoste et al. (2009) Cell Stem Cell 5, 332-342.
-PB introduced neuronal differentiation factor Sox1 gain of function transgene along with loss of function shRNA against pluripotent factors Oct4, Gata6, Sox17, Brachyury or Cdx2
-Neural specific differentiation markers increased in expression and pluripotent marker expression decreases when dox induces PB-mediated differentiation
-Flattened cell types in groups of 10-50 round structures demonstrate neural rosette formation
-Staining of neuroectodermal marker Pax6 and neural precursor nestin confirms neural cell formation
Lacoste et al. (2009) Cell Stem Cell 5, 332-342.
-PB transposase and recipient plasmid are transfected to excise the PB differentiation constructs from the neural cells.
-Following PB-differentiation factor removal, cells remain differentiated as neurites expressing specific surface markers Nestin, DCX, NFH, and Map2
-PCR genotyping confirms that all differentiation factors were excised from the hES cells upon PB transposase re-expression
Lacoste et al. (2009) Cell Stem Cell 5, 332-342.
There are multiple opportunities for licensing piggyBac (PB) cell engineering technology
◦ Research◦ Vector Kits◦ iPS Cell Reprogramming◦ iPS/ES Cell Engineering◦ Cell Engineering◦ Therapeutics
