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Highly efficient and low-mosaicism piggyBac transgenesis platform for rapid founder phenotyping [Amplicon-seq]

ABSTRACT: Pronuclear microinjection is the most widely used method for producing transgenic (Tg) animals. Because its efficiency is typically <20%, phenotypic characterization of Tg animals is usually performed from the next generation (F1) onwards. Conventional Cre-loxP-based conditional knockout (cKO) mouse production requires generating two genetically modified strains and multiple rounds of breeding before cKO mice are available for analysis. Here, we optimized a piggyBac transposon-based method of Tg mouse production and established conditions under which nearly all founder (F0) embryos are transgenic. Using a single-cell RNA sequencing-based strategy, we characterized mosaicism in F0 embryos and demonstrated that piggyBac-mediated transgene integration occurs early in embryonic development. We also achieved ~70% efficiency in generating bacterial-artificial-chromosome-Tg mice. By combining this method with genome editing, we developed a strategy for tissue-specific-knockout phenotyping in the F0 generation. Our findings establish a highly efficient piggyBac-based platform that expands experimental options for Tg animal production by supporting rapid F0-based phenotypic assessment while also facilitating efficient generation of F0 animals for subsequent breeding.

ORGANISM(S): Mus musculus

PROVIDER: GSE308034 | GEO | 2026/06/11

REPOSITORIES: GEO

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Highly efficient and low-mosaicism piggyBac transgenesis platform for rapid founder phenotyping [single-cell RNA-seq]

Project description:Pronuclear microinjection is the most widely used method for producing transgenic (Tg) animals. Because its efficiency is typically <20%, phenotypic characterization of Tg animals is usually performed from the next generation (F1) onwards. Conventional Cre-loxP-based conditional knockout (cKO) mouse production requires generating two genetically modified strains and multiple rounds of breeding before cKO mice are available for analysis. Here, we optimized a piggyBac transposon-based method of Tg mouse production and established conditions under which nearly all founder (F0) embryos are transgenic. Using a single-cell RNA sequencing-based strategy, we characterized mosaicism in F0 embryos and demonstrated that piggyBac-mediated transgene integration occurs early in embryonic development. We also achieved ~70% efficiency in generating bacterial-artificial-chromosome-Tg mice. By combining this method with genome editing, we developed a strategy for tissue-specific-knockout phenotyping in the F0 generation. Our findings establish a highly efficient piggyBac-based platform that expands experimental options for Tg animal production by supporting rapid F0-based phenotypic assessment while also facilitating efficient generation of F0 animals for subsequent breeding.

2026-06-11 | GSE306203 | GEO

Highly efficient and low-mosaicism piggyBac transgenesis platform for rapid founder phenotyping [Transgene-specific scRNA-seq]

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Project description:piggyBac Tg monkey

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Project description:Adult F0 female zebrafish were fed with control diet or diets containing 5-AZA, MeHg or TCDD. After breeding with unexposed males to produce the F1 generation, livers were sampled from the F0 females. F1 generation embryos were unexposed to test chemicals, were sampled, then bred to produce F2 fish, also unexposed to test chemicals. Methylated DNA immunoprecipitation was carried out on liver samples from F0 and F1 and F2 embryos and MeDIP samples were labeled with Cy5 and hybridised to a zebrafish CGI tiling array versus Cy3-labled zebrafish genomic DNA. The objective was to determine DNA methylation changes following chemical exposure and whether these persisted transgenerationally.

2014-03-01 | E-MTAB-2057 | biostudies-arrayexpress

Using a rhabdomyosarcoma patient-derived xenograft to examine precision medicine approaches and model acquired resistance.

Project description:Original patient tumor is directly implanted in mice xenografts. Tumor is propagated to multiple mice for conduct of 6 arm treatment trials and control. Therapies are selected based on T0 and F0 genomic profiles. ICE-T refractory tumor are implanted and additional 6 arm treatment trial and control conducted based on genomic profiles of F2 generation mice to suggested therapies Original patient tumor expanded in F0 mouse generation to F1 generation for multiple arm therapy trial and then refractory tumors implanted for additional multiple arm therapy trial

2014-07-22 | E-GEOD-51130 | biostudies-arrayexpress

IDS transgen arraYS

Project description:Adult female Wistar rats (about 220g) obtained from a breeding colony were mated and fed either a protein sufficient (PS) or protein restricted (PR) diet (n = 6 per dietary group) during F0 pregnancy which provided an increase in energy of approximately 25% compared to the diet fed to the breeding colony (2018S). During lactation dams were fed AIN93G and litters were standardisied to 8 offspring within 24 hours of birth with a bias towards females. Offpsring were weaned onto AIN93M at postnatal day 28 and F1 and F2 females were mated on postnatal day 70 (n = 6 per F0 dietary group). F1 and F2 dams were fed the PS diet during pregnancy and AIN93G during lactation. Offspring were weaned onto AIN93M. On postnatal day 70 unmated female offspring were fasted for 12 hours then sacrificed for hepatic transcritpome analysis by microarray. Expression of 1,684 genes differed by at least 2 fold between adult female F1 offspring of F0 dams from both dietary groups. 1680 genes were altered in F2 offspring and 2,065 genes altered in F3 offspring. Expression of 113 genes was altered in all three generations. Of these, 47% showed directionally opposite differences between generations. Gene ontology analysis revealed clear differences in the pathways altered in each generation. F1 and F2 offspring of F0 dams fed a PR diet showed impaired fasting glucose homeostasis. Hepatic phosphoenolpyruvate carboxykinase (PEPCK) expression was elevated in F1 and F2 offspring from F0 PR dams, but decreased in F3, compared to PS offspring

2011-06-01 | E-MEXP-3205 | biostudies-arrayexpress

Extended abstinence from morphine normalizes sperm small RNA expression and prevents transmission of multigenerational phenotypes

Project description:We examined the impact of paternal abstinence following morphine self_administration on the transmission of multigenerational factors from the F0 to the F1 generation. Previously, we found that offspring of non_abstinent, morphine administering male rats showed increased morphine_taking, diminished adolescent social play, and reduced sensitivity to morphine analgesia. Here, we show that these phenotypes are normalized in the offspring of fathers who were 90 days abstinent from morphine at the time of breeding. We further compared the small RNA content of sperm collected from non_abstinent versus 90 days abstinent F0 males to identify candidate molecules correlated with transmission of morphine_induced multigenerational phenotypes.

2025-05-30 | GSE274906 | GEO

Treatment of TT1 mice with transposon-based gene therapy

Project description:DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems - the only DNA transposons currently employed in clinical trials - during therapeutic intervention, we treated the mouse model of Tyrosinemia type I. with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new Next Generation Sequencing procedure called Streptavidin-Based Enrichment Sequencing, which allowed us to identify approximately 1 million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

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Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites

Project description:Female and male rats of an outbred female Sprague Dawley rats were administered daily intraperitoneal injections of vinclozolin (100 mg/kg BW/day), or DDT (25 mg/kg BW/day) or dimethyl sulfoxide (DMSO) in oil (1 µl/kg BW/day vehicle). Treatment lineages are designated “control” or “vinclozolin” or “DDT” lineages. The gestating female rats treated were designated as the F0 generation. The offspring of the F0 generation rats were the F1. Non-littermate females and males aged 70-90 days from F1 generation of control, vinclozolin or DDT were bred to obtain F2 generation offspring. The F2 generation rats were similarly bred to obtain the F3 generation offspring. Individuals were maintained for 120 days and euthanized for sperm collection. Sperm from multiple individual rats was pooled into single samples. For the control samples, 2 pools were made using 6 animals and 1 pool with 5 animals. For the DDT samples, 3 pools were mady using 4 animals. For the Vinclozolin samples, 3 pools were made using 4 animals. Genomic DNA was isolated and histone chromatin immunoprecipitation with genomic DNA was performed. Each pooled sample received a separate index primer. NGS was performed at the WSU Spokane Genomics Core using Ilumina HiSeq 2500 with a PE50 application, with a read size of approximately 50 bp and approximately 35 million reads per pool.

2018-01-12 | GSE106125 | GEO

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Project description:Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals, and cell types make the cerebellum an excellent model for addressing the critical function of polarity complex proteins in the generation and organization of neural tissues. Deleting apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors (CGNP) in the external granule layer (EGL). In the Pals1 mutants, expression of genes that regulate cell cycle were diminished, correlating with the loss of proliferating population of germinal zones. Furthermore, enhanced Shh signaling through activated Smoothened (Smo) cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a new intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling. Two groups of samples are included: mRNA obtained from 4 WT and 4 CKO animals at E17.5. hGFAP-Cre mice was used to delete Pals1 from most cerebellar neurons and glia (termed Pals1 CKO). Pals1fl/fl and hGFAP-Cre (Zhuo et al., 2001) were bred for generation of CKO.

2016-06-30 | E-GEOD-75696 | biostudies-arrayexpress

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