Reprogramming Mouse Fibroblasts with <i>piggyBac</i> Transposons

Behringer, Richard R.; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, András

doi:10.1101/pdb.prot092627

Cited by 10 publications

(7 citation statements)

References 13 publications

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“…Female C57Bl/6 mice between 3-4 weeks of age were superovulated by intraperitoneal injection of Gestyl followed by human chorionic gonadotropin according to standard procedures (Behringer, 2014). After superovulation, the females were setup with male studs for mating.…”

Section: Methodsmentioning

confidence: 99%

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Mitschka

Mayr

2020

Preprint

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Inactivation of p53 occurs in many cancers. microRNAs and RNA-binding proteins are thought to repress p53 expression through its 3’ untranslated region (3’UTR), thus contributing to tumorigenesis. We used CRISPR/Cas9 to delete the human and mouse p53 3’UTRs while preserving endogenous mRNA processing. This revealed that the endogenous 3’UTR is not involved in the regulation of p53 mRNA or protein expression, neither in steady state nor after genotoxic stress.

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Section: Methodsmentioning

confidence: 99%

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Mitschka

Mayr

2020

Preprint

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“…The methylated and unmethylated ICR-F were microinjected into the paternal or maternal pronucleus, and the methylation status of the ICR-F was analyzed at blastocyst stage. Genomic DNA was extracted from several pools of 5–10 blastocysts, so that each pool contains approximately 1 transgenic blastocyst (the efficiency of transgenesis is known to be ~10% [ 3 ]), and subjected to Dpn I digestion to eliminate the originally-injected DNA. After sodium bisulfite treatment, nested-PCR was conducted with transgene-specific primer sets to amplify DNA sequences covering CTCF1/2 and CTCF3/4 regions, of which methylation status was reported to be involved in regulation of the imprinted expression of H19 and Igf2 [ 4 , 9 ].…”

Section: Resultsmentioning

confidence: 99%

Fate of methylated/unmethylated <i>H19</i> imprinting control region after paternal and maternal pronuclear injection

et al. 2017

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The paternal-allele-specific methylation of the Igf2/H19 imprinting control region (ICR) is established during gametogenesis and maintained throughout development. To elucidate the requirement of the germline passage in the maintenance of the imprinting methylation, we established a system introducing a methylated or unmethylated ICR-containing DNA fragment (ICR-F) into the paternal or maternal genome by microinjecting into the paternal or maternal pronucleus of fertilized eggs, and traced the methylation pattern in the ICR-F. When the ICR-F was injected in a methylated form, it was demethylated approximately to half degree at blastocyst stage but was almost completely remethylated at 3 weeks of age. In the case of the unmethylated form, the ICR-F remained unmethylated at the blastocyst stage, but was almost half-methylated at 3 weeks of age. Interestingly, the paternally injected ICR-F was highly methylated compared with maternally injected ICR-F at 3 weeks of age, partially mimicking the endogenous methylation pattern. Moreover, introduction of mutations in the CTCF (CCCTC binding factor) binding sites of the ICR-F, which are known to be important for the maintenance of hypomethylated maternal ICR, induced hypermethylation of the mutated ICR-F in both paternal and maternal pronuclear injected 3-week-old mice. Our results suggest the presence of a protection-against-methylation activity of the CTCF binding site in establishing the preferential paternal methylation during post-fertilization development and the importance of germline passage in the maintenance of the parental specific methylation at H19 ICR.

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“…Due to efficient cloning strategies, the piggyBac system has readily lent itself for screenings in vivo ( Xu et al, 2017 ), and the possibility of removing the transgene without a footprint by transposase-mediated excision ( Woltjen et al, 2009 ; Behringer et al, 2017 ) is promising for translational approaches. The application of the piggyBac transposon for in vivo studies has been further encouraged by the development of improved transposase enzymes that, based on the original PBase, have been optimized for their use e.g., in mammalian systems.…”

Section: Tools For Stable Expression Of Genes Of Interestmentioning

confidence: 99%

Non-codon Optimized PiggyBac Transposase Induces Developmental Brain Aberrations: A Call for in vivo Analysis

Vierl

Kaur

Götz

2021

Front. Cell Dev. Biol.

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In this perspective article, we briefly review tools for stable gain-of-function expression to explore key fate determinants in embryonic brain development. As the piggyBac transposon system has the highest insert size, a seamless integration of the transposed sequence into the host genome, and can be delivered by transfection avoiding viral vectors causing an immune response, we explored its use in the murine developing forebrain. The original piggyBac transposase PBase or the mouse codon-optimized version mPB and the construct to insert, contained in the piggyBac transposon, were introduced by in utero electroporation at embryonic day 13 into radial glia, the neural stem cells, in the developing dorsal telencephalon, and analyzed 3 or 5 days later. When using PBase, we observed an increase in basal progenitor cells, often accompanied by folding aberrations. These effects were considerably ameliorated when using the piggyBac plasmid together with mPB. While size and strength of the electroporated region was not correlated to the aberrations, integration was essential and the positive correlation to the insert size implicates the frequency of transposition as a possible mechanism. We discuss this in light of the increase in transposing endogenous viral vectors during mammalian phylogeny and their role in neurogenesis and radial glial cells. Most importantly, we aim to alert the users of this system to the phenotypes caused by non-codon optimized PBase application in vivo.

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Reprogramming Mouse Fibroblasts with piggyBac Transposons

Cited by 10 publications

References 13 publications

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Fate of methylated/unmethylated <i>H19</i> imprinting control region after paternal and maternal pronuclear injection

Non-codon Optimized PiggyBac Transposase Induces Developmental Brain Aberrations: A Call for in vivo Analysis

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