Targeted genetic modification of cell lines for recombinant protein production

Barron, Niall; Piskareva, Olga; Muniyappa, Mohan Kumar

doi:10.1007/s10616-007-9050-y

Cited by 6 publications

(7 citation statements)

References 54 publications

(50 reference statements)

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“…These barrier elements include scaffold or matrix attachment regions (S/MARs), insulators, anti‐repressor elements, and ubiquitous chromatin opening elements (UCOEs) (Barnes and Dickson, 2006; Kwaks and Otte, 2006). Alternative approaches to improving productive and stable transgene expression rely on the targeting of highly transcribed sites within the host cell genome by means of heterologous site‐specific recombination (Barron et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Matasci

Baldi

Hacker

et al. 2011

Biotech & Bioengineering

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Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening.

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Section: Introductionmentioning

confidence: 99%

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Matasci

Baldi

Hacker

et al. 2011

Biotech & Bioengineering

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“…Sandström et al demonstrated the ability to bind nucleic acids onto gold nanoparticles [151], and a similar modification has been done by Rosi et al where tetrathiol-modified antisense oligonucleotides were bound to 13-nm gold nanoparticles [152]. Being able to conjugate nucleic acids to nanoparticles opens up the possibility of targeted gene delivery, which could, for example, lead to genes coding for a specific protein to be delivered to a cell that was either deficient in that protein or could not produce the protein themselves [156]. It has also been exhibited that gold nanoparticles modified with DNA can transfect cancer cells [157] (Table 4).…”

Section: Bio-medical Applications Of Metal Nanoparticlesmentioning

confidence: 99%

Magnetic Functionalized Nanoparticles for Biomedical, Drug Delivery and Imaging Applications

2019

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Medicine is constantly looking for new and improved treatments for diseases, which need to have a high efficacy and be cost-effective, creating a large demand on scientific research to discover such new treatments. One important aspect of any treatment is the ability to be able to target only the illness and not cause harm to another healthy part of the body. For this reason, metallic nanoparticles have been and are currently being extensively researched for their possible medical uses, including medical imaging, antibacterial and antiviral applications. Superparamagnetic metal nanoparticles possess properties that allow them to be directed around the body with a magnetic field or directed to a magnetic implant, which opens up the potential to conjugate various bio-cargos to the nanoparticles that could then be directed for treatment in the body. Here we report on some of the current bio-medical applications of various metal nanoparticles, including single metal nanoparticles, functionalized metal nanoparticles, and core-shell metal nanoparticles using a core of Fe 3 O 4 as well as synthesis methods of these core-shell nanoparticles.

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“…Esto puede tener como consecuencia la interrupción de genes importantes en el genoma del hospedero y dar origen a genotipos desconocidos que son incapaces de sintetizar la PR. En el mejor de los casos se puede producir la PR, pero con bajo rendimiento (Barron et al, 2007).…”

Section: Limitaciones En La Producción De Prunclassified

“…Estos errores aleatorios son difíciles de detectar, ya que producen una mezcla heterogénea de polipéptidos. Las proteínas alteradas pueden estar presentes en cantidades muy pequeñas, pero el número total de moléculas defectuosas podría ser sustancial en el rendimiento de la PR (Barron et al, 2007;Mattanovich et al, 2004).…”

Section: Limitaciones En La Producción De Prunclassified

“…Posteriormente se utilizan enzimas de restricción para clonar el ADNc (o fragmento de interés) en un vector de expresión basado T7-ARNpol (sistema pET ® ). En caso de ser necesario, esos sitios únicos de restricción se pueden introducir mediante PCR con iniciadores que se extiendan corriente arriba y abajo de la región codificante de interés (Barron et al, 2007;Geisse y Fux, 2009;Palomares et al, 2004).…”

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Perspectivas Actuales Del Uso De Proteínas Recombinantes Y Su Importancia en La Investigación Científica E Industrial

Guevara-Hernández¹,

López-Zavala²,

Lr³

et al. 2013

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RESUMENEl descubrimiento de la estructura y el mecanismo de replicación del ADN han permitido el desarrollo de técnicas biomoleculares para su manipulación, gracias a estos avances es posible sintetizar proteínas en organismos en los que no se encuentran de manera natural (sobreexpresión heteróloga). A las proteínas producidas de esta manera se les conoce como proteínas recombinantes (PR).Las PR se pueden producir en una gran variedad de sistemas biológicos, como bacterias, levaduras y hasta células eucariontes. Comercialmente están disponibles un gran numero de sistemas de expresión y uno de los más utilizados es el sistema basado en la ARN polimerasa del fago T7. Las PR pueden tener características estructurales y funcionales muy similares a las proteínas naturales y por lo general se producen con alta eficiencia. Sin embargo, algunas PR no son funcionales o sufren problemas de plegamiento cuando se expresan en células procariontes, formando agregados moleculares que se conocen como cuerpos de inclusión. La coexpresión con otras proteínas, como las chaperonas o el uso de cepas modificadas para la sobreexpresión, son algunas de las estrategias utilizadas para evitar la formación de agregados. A pesar de estas limitantes, la tecnología de PR es ampliamente utilizada en investigación y en la industria farmacéutica y alimentaria. Esta revisión tratará sobre los principales aspectos del proceso de producción de PR y sus aplicaciones. ABSTRACTThe discovery of DNA's structure has allowed the development of molecular techniques for its manipulation. With these advances it is possible to synthesize proteins in organisms that are not found naturally (called protein heterologous over expression). A protein produced in this way is known as recombinant proteins (PR), since it is the product of recombinant DNA. The PR can be produced in a variety of systems, like bacteria, yeast and eukaryotic cells. Likewise, there are several expression systems available; being the system based on phage T7 RNA polymerase one of the most used. Usually, PR may have similar structural and functional properties as the natural protein, and they are produced in high yield. However, some PR is difficult to obtain due to the formation and accumulation of aggregates (inclusion bodies). The chaperone co-expression and the use of modified host strains are some strategies used to solve some of these problems. Despite these limitations, PR technology has been widely used in different industries such as pharmaceuticals, food, among others. On the other hand, the use of PR has enabled major advances in the understanding of diverse cellular processes in scientific research.

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Targeted genetic modification of cell lines for recombinant protein production

Cited by 6 publications

References 54 publications

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Magnetic Functionalized Nanoparticles for Biomedical, Drug Delivery and Imaging Applications

Perspectivas Actuales Del Uso De Proteínas Recombinantes Y Su Importancia en La Investigación Científica E Industrial

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