Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids

Liu, Xiaomei; Han, Ping; Zhang, Chun

doi:10.1186/1756-0500-7-626

Cited by 4 publications

(3 citation statements)

References 17 publications

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“…However, random integration may result in incomplete insertion of the destination gene and highly heterogeneous expression levels among cells, clone selection is usually needed. Furthermore, due to low integration frequency, it is quite time-consuming, establishing a stable cell may take up to 6 months [ 30 , 31 ]. CRISPR activates endogenous gene overexpression by transcriptional activation domain like VP64 that fused dCas9.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon

et al. 2021

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Background Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. Results We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3ʹ-end. Consequently, the secondary structures were changed, the RNA–protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3ʹ-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. Conclusions Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study. Graphic Abstract

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Section: Discussionmentioning

confidence: 99%

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon

et al. 2021

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“…This could be achieved through the production of a NHEJ enabled frame-shift mutation causing an otherwise out-of-frame stop codon to be translated. This can be efficiently achieved in livestock as demonstrated by Simon Lillico and colleagues for the porcine RELA gene (Lillico et al 2013). An alternative strategy could be to delete the transcriptional start site and/or the translational AUG codon, thus inactivating the target gene.…”

Section: What Could Be Done – the Opportunitiesmentioning

confidence: 99%

“…To illustrate the potential, identification of the genetic variation controlling lactation may come from comparison of the underlying genetics which controls the extreme phenotypic differences in milk production between Bos taurus and Bos indicus cattle. Precision breeding through the use of genome editors would enable introgression of favourable alleles (as is being attempted for virus resilience in pigs: (Lillico et al 2013). This could have major rewards with regard to Bos indicus milk production in the tropical regions of the world.…”

Section: What Could Be Done – the Opportunitiesmentioning

confidence: 99%

Genetically engineering milk

Whitelaw

Joshi

Kumar

et al. 2016

Journal of Dairy Research

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It has been thirty years since the first genetically engineered animal with altered milk composition was reported. During the intervening years, the world population has increased from 5bn to 7bn people. An increasing demand for protein in the human diet has followed this population expansion, putting huge stress on the food supply chain. Many solutions to the grand challenge of food security for all have been proposed and are currently under investigation and study. Amongst these, genetics still has an important role to play, aiming to continually enable the selection of livestock with enhanced traits. Part of the geneticist's tool box is the technology of genetic engineering. In this Invited Review, we indicate that this technology has come a long way, we focus on the genetic engineering of dairy animals and we argue that the new strategies for precision breeding demand proper evaluation as to how they could contribute to the essential increases in agricultural productivity our society must achieve.

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AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines

Luo¹,

Frederick²,

Martin³

et al. 2017

Human Gene Therapy Methods

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Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

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Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids

Cited by 4 publications

References 17 publications

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon

Genetically engineering milk

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines

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