GEN - Genetic Engineering and Biotechnology News
Feb 1, 2013 (Vol. 33, No. 3)
Tying Up Loose Ends in Cell-Line Development
by Angelo DePalma, Ph.D.
NovelExpression Methodology
Genome-editing technologies now permit precise positioning of deletions, modifications, and
transgenes within living cells. These ideas have led Zsolt Keresztessy, Ph.D.,
senior research fellow at Proxencell, to a method employing sequence-specific
meganucleases and TAL effector nucleases to generate stable monoclonal cell
lines expressing membrane-bound antigens, FCGR receptors, and monoclonal
antibodies. TAL effector nucleases are novel sequence-specific nucleases,
formed by fusing a transcription activator-like (TAL) effector DNA binding domain
to the catalytic head of an endonuclease.
transgenes within living cells. These ideas have led Zsolt Keresztessy, Ph.D.,
senior research fellow at Proxencell, to a method employing sequence-specific
meganucleases and TAL effector nucleases to generate stable monoclonal cell
lines expressing membrane-bound antigens, FCGR receptors, and monoclonal
antibodies. TAL effector nucleases are novel sequence-specific nucleases,
formed by fusing a transcription activator-like (TAL) effector DNA binding domain
to the catalytic head of an endonuclease.
As a core facility for the University of Debrecen in Hungary, Proxencell provides
protein expression services using optimized synthetic genes and expression
organisms that include bacteria, yeast, and both insect and mammalian cells for
small- and large-scale protein production.
protein expression services using optimized synthetic genes and expression
organisms that include bacteria, yeast, and both insect and mammalian cells for
small- and large-scale protein production.
Dr. Keresztessy explains that specific genome editing technologies are still in the
initial evolutionary phase—true especially for TAL effector nucleases. “That
means, in addition to requiring substantial optimization work, investigators
must also innovate in the adaptation of commercially available systems from,
for example, Cellectis Bioresearch or Life Technologies.”
initial evolutionary phase—true especially for TAL effector nucleases. “That
means, in addition to requiring substantial optimization work, investigators
must also innovate in the adaptation of commercially available systems from,
for example, Cellectis Bioresearch or Life Technologies.”
Uncovering effective ways to transfer and express sequence-specific nucleases (e.g.,
plasmid DNA, mRNA, or proteins) into your target cells or cell lines, together
with accessory sequences including like templates for homologous recombination
or genome editing reporter constructs, is critical.
plasmid DNA, mRNA, or proteins) into your target cells or cell lines, together
with accessory sequences including like templates for homologous recombination
or genome editing reporter constructs, is critical.
“As a result, we were forced to develop new technologies for assessing genome
modifications at early stages of TAL transfections, strategies and tools for
detecting and enriching knockout cells, and new approaches for mapping TAL
specificity in vivo in automated and high-throughput assays.”
modifications at early stages of TAL transfections, strategies and tools for
detecting and enriching knockout cells, and new approaches for mapping TAL
specificity in vivo in automated and high-throughput assays.”
Dr. Keresztessy and colleagues have enjoyed several successes in designing
bioassay-worthy cells through these strategies. More relevant here are
production cells. When the goal is overexpressing a protein—for example, a
monoclonal antibody—for large-scale production, a variety of choices exist from
commercially available reagent kits. Dr. Keresztessy’s system of choice is the
cGPS CHO-Sa CEMAX system from Cellectis.
bioassay-worthy cells through these strategies. More relevant here are
production cells. When the goal is overexpressing a protein—for example, a
monoclonal antibody—for large-scale production, a variety of choices exist from
commercially available reagent kits. Dr. Keresztessy’s system of choice is the
cGPS CHO-Sa CEMAX system from Cellectis.
“However, we needed to construct our own version of the integration matrix vector
provided by the vendor to efficiently express our mutant therapeutic
antibodies, which we aimed to use as cellular assay controls in our research
applications. Into the vector, we cloned the synthetic genes of the light and
heavy chains of a human IgG framework, with the desired mutations, linked via
an in-house designed and optimized IRES sequence.”
provided by the vendor to efficiently express our mutant therapeutic
antibodies, which we aimed to use as cellular assay controls in our research
applications. Into the vector, we cloned the synthetic genes of the light and
heavy chains of a human IgG framework, with the desired mutations, linked via
an in-house designed and optimized IRES sequence.”
DEVELOPING IMMUNOPRECIPITATION/NEXT GENERATION SEQUENCING-BASED TECHNOLOGIES AND TOOLS FOR
ANTIBODY CHARACTERISATION:
ANTIBODY CHARACTERISATION:
Specificity,
batch-to-batch QC, and full process controls for research, therapeutic and
diagnostic applications
batch-to-batch QC, and full process controls for research, therapeutic and
diagnostic applications
Zsolt
Keresztessy1,2, Attila Horváth2, Ádám Pallér2, László Steiner2, József
Horváth1, Gábor Zahuczky2, László Nagy2, 3& Bálint L. Bálint2,3,
Keresztessy1,2, Attila Horváth2, Ádám Pallér2, László Steiner2, József
Horváth1, Gábor Zahuczky2, László Nagy2, 3& Bálint L. Bálint2,3,
1Protein
Expression and Cell Engineering Laboratory, 2Center for Clinical Genomics and
Personalised Medicine,
Expression and Cell Engineering Laboratory, 2Center for Clinical Genomics and
Personalised Medicine,
3Department
of Biochemistry and Molecular Biology, Medical and Health Sci Center,
University of Debrecen
of Biochemistry and Molecular Biology, Medical and Health Sci Center,
University of Debrecen
PROXENCELL started a recombinant antibody production development project.
PROXENCELL initiated cell/genome engineering projects for stable cell line development
DCGC presents a poster on PEGS 2011 in Boston, USA.