A Novel Method for Removing Polyethyleneimine from Biopharmaceutical Samples: Improving Assay Sensitivity of Residual DNA Qpcr

Zhang, Shumin; Roberts, Matthew; Jones, Marisa; Zeitler, Jacob; Kilby, Greg W.; Liu, Aston; White, John R.

doi:10.2144/btn-2020-0011

Cited by 2 publications

(1 citation statement)

References 13 publications

(15 reference statements)

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“…To assess a possible molecular basis for herbicide resistance, the ALS genes from three shattercane biotypes, P2-205, P8-30, and P9-102, showed resistance to primisulfuron and wild-type shattercane (susceptible) and were sequenced and compared. Sequencing primers were designed based on the corn sequence [41] (Table 6). They were located at approximately 500-bp intervals inforward and reversed orientation, providing 2-to fourfold coverage of the ALS gene.…”

Section: Dna Extraction Amplification and Sequencing Of The Als Genementioning

confidence: 99%

Mutations in the acetolactate synthase (ALS) enzyme affect shattercane (Sorghum bicolor) response to ALS-inhibiting herbicides

et al. 2023

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Background Shattercane [Sorghum bicolor (L.) Moench ssp. Arundinaceum (Desv.)] is a competitive weed in North America's corn, soybean, sorghum, and other agronomic crops. Control of shattercane with POST herbicides in corn became possible with the introduction of acetolactate synthase (ALS)-inhibiting herbicides in the 1980s, and their extensive use resulted in the evolution of ALS-inhibitors resistant shattercane. Results Shattercane seeds were collected from 16 south-eastern and south-central Nebraska fields that were treated with primisulfuron for three consecutive years. Three resistant plants were found in greenhouse evaluations of more than 30,000 plants. Results from a greenhouse bioassay conducted to assess the response of each shattercane biotype to ALS-inhibiting herbicides showed a differential response to ALS inhibitors within and between chemical classes. Biotype P8-30 was resistant or partially resistant to all ALS-inhibiting herbicides applied and displayed a unique amino acid sequence substitution (Trp574 to Leu) relative to the other two resistant biotypes, P2-205 and P9-102. Whole plant dose–response studies confirmed a 4- to the 12-fold level of primisulfuron resistance in three shattercane biotypes compared with the known primisulfuron-susceptible shattercane biotype. The ALS gene was sequenced using primers designed from the corn ALS sequence to identify mutations in the ALS gene that confer resistance. A total of seven nucleotide substitutions were detected in the three herbicide-resistant biotypes P2-205, P8-30, and P9-102. These biotypes are being crossed to adapted sorghum lines (grain, sweet, and forage) to broaden germplasm with resistance to ALS-inhibiting herbicides. Conclusion The discovery of these mutants should accelerate the development of sorghum genotypes that tolerate ALS-based herbicides, which provide additional choices for sorghum farmers to control weeds, especially grasses, in their fields.

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Section: Dna Extraction Amplification and Sequencing Of The Als Genementioning

confidence: 99%

Mutations in the acetolactate synthase (ALS) enzyme affect shattercane (Sorghum bicolor) response to ALS-inhibiting herbicides

et al. 2023

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Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno‐associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids

Srinivasan,

Canova,

Sha

et al. 2024

Biotech & Bioengineering

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Recombinant adeno‐associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long‐term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled‐to‐total capsids (~1%–30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid‐filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.

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A Novel Method for Removing Polyethyleneimine from Biopharmaceutical Samples: Improving Assay Sensitivity of Residual DNA Qpcr

Cited by 2 publications

References 13 publications

Mutations in the acetolactate synthase (ALS) enzyme affect shattercane (Sorghum bicolor) response to ALS-inhibiting herbicides

Mutations in the acetolactate synthase (ALS) enzyme affect shattercane (Sorghum bicolor) response to ALS-inhibiting herbicides

Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno‐associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids

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