Check patentability & draft patents in minutes with Patsnap Eureka AI!

Transduction buffer

a technology of transduction buffer and buffer, which is applied in the direction of peptides, polypeptides with his-tags, enzymology, etc., can solve the problems of inability to demonstrate the release of trypan blue into the cytoplasm or other cellular structures, the lack of reliable, non-toxic and efficient methods to achieve this effect, and the cell membrane presents a major obstacle to the introduction of many biologically active molecules

Pending Publication Date: 2021-03-25
KONINK NEDERLANDSE AKADE VAN WETENSCHAPPEN
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The method enables robust and efficient introduction of small and macromolecules into primary cells and stem cell lines, improving transduction efficiency and cell viability, and can be used for genetic modification and therapeutic applications.

Problems solved by technology

Unfortunately, the cell membrane presents a major obstacle for the introduction of many biologically active molecules.
While the ability to introduce proteins into cells has many applications in research and medicine, a reliable, non-toxic and efficient method to do so is still lacking.
However, the authors did not demonstrate that the trypan blue was released into the cytoplasm or other cellular compartments following uptake.
Furthermore, trypan blue is a small molecule and it was not clear it the procedure would allow uptake of macromolecules as well.
As such, the method described by Castellot and colleagues cannot be applied for the transduction of macromolecules into primary cells and / or stem cell lines.
Unfortunately, this technique proved limited to immortalized cell lines, and yields poor protein transduction efficiencies in primary cells.
However, the strong positive charge of the TAT peptide severely hampers the production of native recombinant TAT-fusion proteins in E. coli, with much of the recombinant protein ending up in inclusion bodies.
In addition, this technology requires the TAT peptide to be fused to the recombinant protein and therefore limits the type and number of proteins that can be transduced.
The TAT peptide itself can disrupt the function or localization of the recombinant protein leading to unexpected or unwanted results.
This application explains that transfection efficiency of “naked” DNA is low.
These methods result in significant risk of adverse reactions, including acute immune rejection due to the high dose of injected virus and tumor formation resulting from viral integration position effects.
Furthermore, the nucleic acid is expressed within the cell for several days resulting in high expression of enzymes and greater likelihood of off-target effects, e.g. genetic modification of non-target sequences within the cell.
Viral transduction also remains inefficient for certain cell types.
These difficulties hamper clinical application of the gene editing systems mentioned above.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Transduction buffer
  • Transduction buffer
  • Transduction buffer

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0404]The ability to introduce small- or macromolecules into cells finds important applications in research and medicine. Unfortunately, the cell membrane presents a major obstacle for the introduction of many biologically active molecules. Significant effort has capitalized on the introduction of nucleotides (DNA, RNA, siRNA) and / or therapeutic molecules into cells, and while primary cells still pose a challenge, progress has been made using cationic lipids, nanoparticles or viral vectors as transmembrane carriers. In comparison, the development of new technologies for the intracellular delivery of proteins has been at a virtual standstill. Nonetheless, the ability to introduce proteins into cells would have many applications in vaccine development, genome editing, epigenetic reprogramming, (stem) cell differentiation and the manipulation of intracellular processes. The development of better technologies for the efficient intracellular delivery of proteins and other macromolecules,...

example 2

CRE Transduction and Quantification of CRE Incorporation

[0551]MEFs were transduced with CRE following the protocol 3 / 700 in a 96 well format. In brief, 12,000 Lox-RFP / STOP-Lox-eGFP MEF cells were seeded per well in gelatin coated plates using MEF media. Next day, CRE protein in 5× transduction buffer was diluted 1 / 5 with DMEM phenol-red free without antibiotics. The complete mixture was then added to cells. After 3 hrs. of transduction, media was replaced by fresh mES media and incubated for 24-48 hrs. Cells were then analyzed by measuring of green and red signal in a fluorescent microscope or in a flow cytometer.

[0552]mES cells were transduced with CRE following the protocol 12 / 500 in a 96 well format. In brief, 75,000 Lox-STOP-Lox-GFP mES cells per well were seeded gelatin coated plates using mES media.

[0553]Next day, Cre protein in 5× transduction buffer was diluted 1 / 5 with mES transduction media. The complete mixture was then added to cells. After 12 hrs. the transduction media...

example 3

TALEN Transduction

[0556]TALEN proteins were expressed and purified under native conditions as described above. The recombinant TALEN proteins were purified in 5× transduction buffer. Human ES cells were transduced with TALEN proteins using the transduction protocol 12 / 500. For HPRT gene disruption, a pair of TALEN proteins targeting the HPRT gene was used. 4 days after the transduction, 2.5 μM 6-TG was added to select for HPRT knockout cells. Two weeks later, surviving clones were picked and expanded for genomic DNA purification and HPRT gene sequencing.

[0557]Male human iPS cells were transduced with 2 uM TALEN protein for 12 hs. In brief, 20 ul HPRT talen proteins in 5× transduction buffer were mixed with 80 ul of human iPS cell media. Final mixture was added to cell for 12 hs. After that, media was replaced by 150 ul of human iPS cell culture media. After 5 days 3 uM 6-TG was added into culture media to select HPRT deficient cells. After 10 days, individual clones were picked and ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
osmolalityaaaaaaaaaa
diametersaaaaaaaaaa
timeaaaaaaaaaa
Login to View More

Abstract

The invention relates to transduction compounds, buffers and methods for introducing molecules into cells. The invention also relates to methods of treatment, pharmaceutical compositions and other uses of the transduction compounds and buffers. The invention also relates to modified cells obtainable by the transduction compounds, buffers and methods of the invention.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 14 / 914,528, filed Feb. 25, 2016, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT / IB2014 / 064127 with an international filing date of Aug. 28, 2014, which claims the benefit of United Kingdom Patent Application No. 1315321.8, filed Aug. 28, 2013, the entire contents of each of which are incorporated by reference herein.TECHNICAL FIELD[0002]This invention relates to transduction buffers and methods for introducing molecules into cells.BACKGROUND ART[0003]The ability to introduce small- or macromolecules into cells finds important applications in research and medicine. Unfortunately, the cell membrane presents a major obstacle for the introduction of many biologically active molecules.[0004]While the ability to introduce proteins into cells has many applications in research and medicine, a reliable, non-toxic and efficient method to do so is still lacking...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86A61K35/545C12N15/87A61K35/30A61K38/17A61K38/45A61K38/46A61K47/10A61K47/12A61K47/18A61K47/20A61K47/22C07K14/32C07K14/47C12N7/00C12N9/12C12N9/16
CPCC12N15/86C12Y301/00C12N15/87A61K35/30A61K38/1709A61K38/45A61K38/465A61K47/10A61K47/12A61K47/18A61K47/20A61K47/22C07K14/32C07K14/4702C12N7/00C12N9/1241C12N9/16C07K2319/21C07K2319/50C07K2319/55C12N2740/15043C12Y207/07A61K35/545A61K31/00
Inventor GEIJSEN, NIELSD'ASTOLFO, DIEGO SEBASTIÁN
Owner KONINK NEDERLANDSE AKADE VAN WETENSCHAPPEN
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com