Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Dhiman, Heena; Campbell, Marguerite; Melcher, Michael; Smith, Kevin D.; Borth, Nicole

doi:10.1016/j.csbj.2020.11.008

Cited by 19 publications

(31 citation statements)

References 42 publications

(50 reference statements)

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“…The two LC copies were located next to each other in a head to tail arrangement within the intended integration site. Previous groups have reported concatemerization of plasmid at a given integration site and demonstrated that the structural variation in the transgene integration region will impact expression, stability, and may contribute to potential rearrangements 25,26 . Our study indicates phenomenon unique to this, as only a specific portion of the vector (LC) was duplicated and was localized in the middle of the vector payload within the SSI landing pad.…”

Section: Discussionmentioning

confidence: 55%

“…Previous groups have reported concatemerization of plasmid at a given integration site and demonstrated that the structural variation in the transgene integration region will impact expression, stability, and may contribute to potential rearrangements. 25,26 Our study indicates phenomenon unique to this, as only a specific portion of the vector (LC) was duplicated and was localized in the middle of the vector payload within the SSI landing pad. Furthermore, the rearrangement is specific to the intended integration site and no offtarget events were observed.…”

Section: Evaluation Of 2xlc Transfection Pools Expressing the 045 Bispecific In An Ssi Expression Systemmentioning

confidence: 80%

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Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule

Tevelev

Patel

Shields

et al. 2021

Biotechnol Progress

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Site specific integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for traditional monoclonal antibodies. As complex modalities such as antibody-like molecules comprised of greater than two peptides become more prevalent, greater emphasis needs to be placed on the ability to produce appreciable quantities of the correct product of interest (POI). The ability to screen several transcript stoichiometries could play a large role in ensuring high amounts of the correct POI. Here we illustrate implementation of an SSI expression system with a single site of integration for development and production of a multichain, bi-specific molecule. A SSI vector with a single copy of all of the genes of interest was initially selected for stable Chinese hamster ovary transfection. While the resulting transfection pools generated low levels of the desired heterodimer, utilizing an intensive clone screen strategy, we were able to identify clones having significantly higher levels of POI. In-depth genotypic characterization of clones having the desirable phenotype revealed that a duplication of the light chain within the landing pad was responsible for producing the intended molecule. Retrospective transfection pool analysis using a vector configuration mimicking the transgene configuration found in the clones, as well as other vector configurations, yielded more favorable results with respect to % POI. Overall, the study demonstrated that despite the theoretical static nature of the SSI expression system, enough heterogeneity existed to yield clones having significantly different transgene phenotypes/genotypes and support production of a complex multi-chain molecule.

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Section: Discussionmentioning

confidence: 55%

Section: Evaluation Of 2xlc Transfection Pools Expressing the 045 Bispecific In An Ssi Expression Systemmentioning

confidence: 80%

Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule

Tevelev

Patel

Shields

et al. 2021

Biotechnol Progress

View full text Add to dashboard Cite

show abstract

“…Recent reports have revealed that integrating genetic materials can change a transcriptional profile due to chromatin and chromosome conformation changes 90 , 91 . In the current study, GCaMP3-KI cells may have the different transcriptional profile compared to wild type cells, which might induce intracellular Ca 2+ surge (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells

Miyata

Fuse

Tokumoto

et al. 2021

Sci Rep

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Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.

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“…However, although unreliable for an accurate CN assessment, this method can clearly demonstrate the presence of T-DNA truncations (due to T-DNA border trimming or of tandem/inverted repeats), thus confirming potential hypothesis formulated on the basis of qPCR and ddPCR results, such as the case of line 3 where inconsistency in the CN of two exogenous genes was caused by a T-DNA truncation. These kinds of rearrangements may have a profound effect on inactivation or variable expression of the transgene [46,47], and their detection is, therefore, crucial. Head-to-tail arrays of multiple copies frequently result in stable expression, whereas head-to-head or tail-to-tail arrangements generally result in silencing [48].…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the location of the integration point may also give indications about the possible influence of the surrounding genomic sequence on recombinant DNA expression, a feature that is known as position effect [49,50]. In recent studies [47,51], random transgene integration sites were analyzed in Chinese hamster ovary cells, mammalian cell lines used as biofactories for the production of therapeutic molecules. These authors found that transgene stability is ensured over time when integration occurred in genomic regions with high transcriptional activity and accessibility to transcription factors (not necessarily within highly transcribed genes).…”

Section: Discussionmentioning

confidence: 99%

Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

Costa

Vinciguerra

Giacomelli

et al. 2021

Eur Food Res Technol

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Agrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.

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Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Abstract: Graphical abstract

Cited by 19 publications

References 42 publications

Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule

Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule

Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells

Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

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