PTSelect™: A post-transcriptional technology that enables rapid establishment of stable CHO cell lines and surveillance of clonal variation.
PTSelect™: A post-transcriptional technology that enables rapid establishment of stable CHO cell lines and surveillance of clonal variation.
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DOI：
10.1016/j.jbiotec.2020.09.025
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发表时间：
2021-01-10
影响因子：
4.1
通讯作者：
Sharfstein ST
中科院分区：
文献类型：
--
作者：
Muralidharan-Chari V;Wurz Z;Doyle F;Henry M;Diendorfer A;Tenenbaum SA;Borth N;Eveleth E;Sharfstein ST
目前，通过抗生素和/或代谢选择建立了稳定的中国仓鼠卵巢细胞系，其产生治疗性重组蛋白。在这里，我们报告了一种新的技术，PTSelect™，其利用克隆在感兴趣的基因（GOI）上游的siRNA，该基因被加工以产生功能性PTSelect™-siRNA，这使得细胞富集成为可能。选择具有稳定整合的GOI的细胞，并通过抑制由PTSelect™-siRNA调节的CD 4/siRNA mRNA并利用细胞表面上CD 4的可变表达将其与不具有GOI的细胞分离。本研究描述了PTSelect™原理，并将PTSelect™开发的克隆的生产率、倍增时间和稳定性与常规开发的克隆进行了比较。PTSelect™快速建立了与传统方法产生的池具有可比稳定性和生产率的池群，并且可以进一步用于容易地监测由于克隆漂移引起的生产率变化，识别生产率降低的单个细胞。
Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here, we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell enrichment. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.
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影响因子：
3.8
作者：
Droz, Xuan;Harraghy, Niamh;Mermod, Nicolas
通讯作者：
Mermod, Nicolas
影响因子：
1.7
作者：
Frye, Christopher;Deshpande, Rohini;Anderson, Karin
通讯作者：
Anderson, Karin
影响因子：
5.3
作者：
Li, Feng;Vijayasankaran, Natarajan;Amanullah, Ashraf
通讯作者：
Amanullah, Ashraf
DOI：
10.1007/978-1-4939-6972-2_9
发表时间：
2017-01-01
期刊：
HETEROLOGOUS PROTEIN PRODUCTION IN CHO CELLS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Gallagher, Clair;Kelly, Paul S.
通讯作者：
Kelly, Paul S.
影响因子：
4.7
作者：
Bort, Juan A. Hernandez;Stern, Beate;Borth, Nicole
通讯作者：
Borth, Nicole