Single Copy Transgene Integration in a Transcriptionally Active Site for Recombinant Protein Synthesis

Biotechnology Journal
Sofie A O'BrienWei-Shou Hu

Abstract

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor re...Continue Reading

References

Dec 18, 1998·The Journal of Biological Chemistry·X LiS R Kain
Sep 19, 2000·Genesis : the Journal of Genetics and Development·M LauthM Meyer
Aug 31, 2002·Cell·Astrid R W SchröderFrederic Bushman
Aug 5, 2003·Genes & Development·Jill A Fahrner, Stephen B Baylin
Nov 5, 2004·Cellular and Molecular Life Sciences : CMLS·X Wu, S M Burgess
Feb 24, 2006·Biotechnology and Bioengineering·Louise M BarnesAlan J Dickson
Feb 27, 2007·Cell·Bing LiJerry L Workman
Mar 14, 2007·Journal of Immunological Methods·Ying HuangBei Fen Shen
Oct 2, 2007·Current Opinion in Biotechnology·Dagmar WirthHansjörg Hauser
Dec 18, 2007·Current Opinion in Biotechnology·Gargi SethWei-Shou Hu
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Aug 19, 2010·Nature Reviews. Genetics·Fyodor D UrnovPhilip D Gregory
Jan 19, 2011·Journal of Molecular Biology·Soeren TuranJuergen Bode
Feb 14, 2012·Biochimica Et Biophysica Acta·Rodolfo GhirlandoGary Felsenfeld
Jul 27, 2012·Biotechnology and Bioengineering·Sarah L DaviesDavid C James
Oct 9, 2012·Biotechnology and Bioengineering·Sandra CristeaGregory J Cost
Nov 22, 2012·Nature Reviews. Molecular Cell Biology·J Keith Joung, Jeffry D Sander
Oct 11, 2013·Biotechnology and Bioengineering·Nandita VishwanathanWei-Shou Hu
Mar 4, 2014·Nature Biotechnology·Jeffry D Sander, J Keith Joung
Mar 13, 2014·Nature Reviews. Genetics·Daria ShlyuevaAlexander Stark
Dec 5, 2014·Epigenetics & Chromatin·Maria Tsompana, Michael J Buck
Jan 7, 2015·Current Protocols in Molecular Biology·Jason D BuenrostroWilliam J Greenleaf
Jan 27, 2015·Journal of Bioscience and Bioengineering·Takanori InaoMasamichi Kamihira
Feb 26, 2015·Scientific Reports·Jae Seong LeeHelene Faustrup Kildegaard
May 15, 2015·Biotechnology Letters·Huong LeWei-Shou Hu
Dec 1, 2015·Biotechnology and Bioengineering·Sowmya BalasubramanianFlorian M Wurm
Jan 24, 2016·BMC Biotechnology·Nathalie VeithStella Marie Reamon-Buettner

❮ Previous
Next ❯

Citations

Aug 26, 2018·Biotechnology and Bioengineering·Arpan A BandyopadhyayWei-Shou Hu
Oct 3, 2018·Biotechnology Journal·Nicole Borth, Wei-Shou Hu
Feb 9, 2020·Biotechnology Progress·Sofie A O'BrienWei-Shou Hu
Oct 24, 2020·Biotechnology and Bioengineering·Stephen BevanPeter M O'Callaghan
Feb 2, 2021·Biotechnology and Bioengineering·Zion LeeWei-Shou Hu
Aug 14, 2020·ACS Synthetic Biology·Joseph R BradyJ Christopher Love
Feb 27, 2019·ACS Synthetic Biology·Nuša PristovšekHelene Faustrup Kildegaard
Oct 10, 2019·ACS Synthetic Biology·Christopher S StachMichael Smanski
Dec 23, 2021·Biotechnology and Bioengineering·Anna StadermannIngo H Gorr

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.