Unknown,Transcriptomics,Genomics,Proteomics

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High-frequency generation of fertile transgenic mice from H19-Igf2 engineered androgenetic haploid embryonic stem cells

ABSTRACT: Recent success in the derivation of mouse haploid embryonic stem cells from androgenetic blastocysts (ahESCs) has provided new avenues for the generation of genetically modified animals. However, the efficiency to produce viable transgenic mice via intracytoplasmic ahESCs injection (ICAI) was very low, which may correlate with the aberrant regulation of imprinted genes. Here we designed to delete the paternal imprinted gene H19 by CRSPR-Cas9 system combined with homologous recombination. The H19 deleted (H19Î) ahESCs maintained haploidy and genome integrity, expressed pluripotency markers, differentiated into embryoid bodies (EBs), and contributed to chimeras after blastocyst injection. These cells exhibited similar imprinting features with sperm cells, and can produce fertile progenies after ICAI at a high efficiency. More importantly, it is feasible to perform genetic manipulations in H19Î ahESCs, and the genomic modifications can be properly transmitted to offspring. Our study will benefit the reproductive medicine in curing the hereditary genetic diseases and infertility in the future. The copy number variations of the two H19Î ahESC lines were analyzed by the SurePrint G3 Mouse CGH 4Ã180 K microarrays (Agilent). Wild-type OG-3 ahESCs were used as reference.

ORGANISM(S): Mus musculus

SUBMITTER: Meili Zhang

PROVIDER: E-GEOD-67563 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abstract: Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell Syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 ICR (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects (VSDs) and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix (ECM) and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis. Methods: E12.5 hearts were lysed with Collagenase, Dispase II, and DNase I. Cardiac endothelial cells were collected using MACS CD31 microbeads and RNA was isolated using RNeasy Micro kit. After confirming RNA integrity using Bioanalyzer, mRNA library was generated from 25ng RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and Ultra II RNA Library Prep Kit. Library quality was assessed by Bioanalyzer and TapeStation. Sequencing was performed on NovaSeq 6000. Quality of raw fastq reads was assessed using FastQC version 0.11.5. Reads were aligned to the GRCm38/mm10 reference using STAR version 2.4.0i with default parameters and maximum fragment size of 2000 bp. Properly paired primary alignments were retained for downstream analysis using Samtools version 1.9. Count matrices were generated using FeatureCounts version 1.6.2 against RefSeq gene annotation and read into DESeq2 to perform normalization and statistical analysis.

Project description:It is increasingly being recognized that physical interactions between distant chromosomal loci influence the expressivity of the genome. Thus, multiple enhancers may converge on a single gene promoter 1,2 and a single enhancer can stochastically loop to multiple promoters 3. Regulatory elements from neighboring domains or from other chromosomes may interact to generate chromatin structures poised for transcription4-8 or repression 9-11. However, a broader perspective on the formation and function of such networks remains largely unexplored. Using the high-resolution 4C (Circular Chromosome Conformation Capture) analysis, we identify here a genome-wide physical network between genomically imprinted domains impinging H19 imprinting control region (ICR). Sequences interacting with the H19 ICR constitutively map at or within most of known imprinted domains both in mouse embryonic stem cells and in vitro-derived embryoid bodies, despite distinct differences in chromatin environments. Moreover, all-to-all 3D DNA FISH analyses revealed that imprinted domains from 7 different chromosomes co-localize with each other in pair-wise patterns, highlighting the dynamic nature of these interactions. The observed interaction network is dependent on CTCF binding sites within the maternally inherited H19 ICR allele and delays reprogramming of epigenetic features at imprinted domains during male germline development. As other imprinted regions can interact with each other in an H19 ICR-dependent manner, the H19 ICR emerges as an organizer of chromosomal networks by specifically recognizing and reconfiguring imprinted domains. We propose that an ancient key regulatory region, such as the H19 ICR, gradually impacted epigenetic modifications via long-range interactions in the germline to facilitate the recruitment of imprinted genes and their formation in clusters during mammalian radiation. Furthermore, the perspective of epigenetic lesions de-stabilizing such chromosomal networks may advance our understanding of how quantitative traits influence human diseases, such as cancer.

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High-frequency generation of fertile transgenic mice from H19-Igf2 engineered androgenetic haploid embryonic stem cells

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