Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice

Wen, Jinkun; Wu, Jinni; Cao, Tianqi; Zhi, Shengyao; Chen, Yuxi; Aagaard, Lars; Zhen, Peilin; Huang, Yanming; Zhong, Jun; Huang, Junjiu

doi:10.1007/s11248-020-00224-9

Cited by 5 publications

(5 citation statements)

References 38 publications

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“…This examination was performed using the qRT-PCR method implemented on selected YhFF#8-Cas9-GFP-PuroR cells, and the results showed that Cas9 was expressed at 50% of the level of GFP and the GAPDH housekeeping gene. The performance values obtained in this study are consistent with those of other studies in which the EF-1-α promoter outperformed the CMV promoter by 40-60% (27,31).…”

Section: Discussionsupporting

confidence: 91%

“…The genetically modified YhFF#8 expressing Cas9 gene resistance gene (shown in Figure 1A). CMV is a promoter derived from cytomegalovirus, while EF-1-α is a eukaryotic and human promoter (26,27). Although measures have been taken to ensure the proper function of these promoters, some reports have indicated the silencing of genes transferred by pseudo-virus vectors after a certain number of cell divisions (starting from the 4 th wk of transduction) (28)(29)(30).…”

Section: Discussionmentioning

confidence: 99%

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The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report

Soheilipour,

Boozarpour,

Aghaei

et al. 2024

IJRM

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Background: Stable Cas9 (CRISPR-associated protein 9)-expressing cell lines have emerged as valuable tools in genetic research, enhancing the efficiency of the CRISPR/Cas9 system and streamlining gene editing procedures. These cell lines enable simultaneous editing of multiple genes and reduce the overall editing time. Objective: This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes. Materials and Methods: Human embryonic kidney 293LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch 8 (YhFF#8) cells were targeted for genetic modification. Transfection of human embryonic kidney 293LTV cells with pCDH-Cas9 plasmid DNA generated pseudo-viral particles. YhFF#8 cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas9 gene were determined by quantitative polymerase chain reaction. Results: The study validated the fidelity of the Cas9 gene cassette sequence and its transcriptional activity. Transduced YhFF#8 cells exhibited green fluorescence, with antibiotic selection resulting in nearly 100% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#8-Cas9-GFP-PuroR cells using fluorescence microscopy. Conclusion: YhFF#8-Cas9-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research. Key words: Fibroblasts, Cell line, Genetic transduction, CRISPR-Cas9.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report

Soheilipour,

Boozarpour,

Aghaei

et al. 2024

IJRM

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“…While Tfap2c was introduced into Col1A1 locus, the remaining factors were delivered into the genome by lentiviral transduction thereby being random in site and number of integrations. Further variations might result from methylation-induced transgene silencing which was shown to occur with transgenes delivered by lentiviral vectors albeit less frequently than following simple retrovirus transduction (Pfeifer et al, 2002;Wen et al, 2021).…”

Section: Tx Medium Promotes Esc To Tsc Conversionmentioning

confidence: 99%

Choice of factors and medium impinge on success of ESC to TSC conversion

et al. 2020

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“…Cell culture experiments aiming at high gene expression employ transposable elements to achieve stable single-copy integrations or induce high-copy amplification in combination with measures to avoid RIGS. These measures may include chromatin relaxation by inhibitors (trichostatin A, butyrate [ 119 ]; 5-azacytidine (5-AzaC) [ 128 ] or attaching functional elements to the transgene (BAC genomic regions, matrix attachment regions (MARs)) [ 12 , 109 , 129 , 130 ].…”

Section: Repeat-induced Gene Silencingmentioning

confidence: 99%

Concatenation of Transgenic DNA: Random or Orchestrated?

Smirnov

Battulin

2021

Genes

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Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.

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Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice

Cited by 5 publications

References 38 publications

The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report

The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report

Choice of factors and medium impinge on success of ESC to TSC conversion

Concatenation of Transgenic DNA: Random or Orchestrated?

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