Epigenetic Events and Germ Line Transmission in Rag1 knock-out Chimeric Mice Produced by CRISPR/Cas9 Technology

Abstract Considering the effects of epigenetic changes on the embryonic development through altering the gene expression of pluripotency, trophectoderm, and imprinting genes, we determined the pattern of epigenetic alterations in transgenic embryos produced by injecting transgenic mESCs into the morula. To aim this, RAG1 knock-out mESCs were produced using CRISPR-Cas9 editing system and then microinjected into morulas to develop chimeric embryos. Afterward, the expression of pluripotency, trophectoderm genes, and imprinting genes in these embryos were evaluated using real-time PCR. Immunohistochemical analysis was carried out to determine the methylation rates of H3K9me3 and H3K4me3. In the following, since epigenetic alterations can be at least one of the possible reasons of male infertility and the loss of germ-line transmission capacity, the germ-line transmission ability in chimeric mice was also evaluated by breeding them and subsequent backcrosses to wild type strains. Our findings showed that the methylation rates of H3K4me3 and H3K9me3 were significantly lower and higher respectively in the RAG1 knock-out embryos compared others groups. Moreover, the chimeric embryos exhibited the decreased expression of NANOG, OCT4, CDX2, TEAD4, and H19 genes. Following the breeding of chimeric males with normal female mouse, 40% of chimeras had no germ-line transmission (GLT), and 60% were infertile.We showed that the manipulation of mESC by CRISPR-Cas9 approach remarkably changed the methylation status of H3K9me3 and H3K4me3, resulting in impaired development of embryos through dysregulation of genes involved embryonic development and then, may be one of the reasons of infertility and lack of GLT.

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Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6755-6758.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6755-6758

Author(s):

B R Stanton ◽

S W Reid ◽

L F Parada

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Embryonic Development ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6755 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6755-6758 ◽

Cited By ~ 23

Author(s):

B R Stanton ◽

S W Reid ◽

L F Parada

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Embryonic Development ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Germ Line Transmission

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Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus

10.1101/2021.05.10.441399 ◽

2021 ◽

Author(s):

Yuji Matsuoka ◽

Taro Nakamura ◽

Takahito Watanabe ◽

Austen A. Barnett ◽

Sumihare Noji ◽

...

Keyword(s):

Germ Line ◽

Fruit Fly ◽

Model Organisms ◽

Induced Mutations ◽

Gryllus Bimaculatus ◽

Knock Out ◽

Fluorescent Marker ◽

Hemimetabolous Insect ◽

Targeted Nucleases ◽

Germ Line Transmission

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

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Maternal germ-line transmission of mutant mtDNAs from embryonic stem cell-derived chimeric mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.250491597 ◽

2000 ◽

Vol 97 (26) ◽

pp. 14461-14466 ◽

Cited By ~ 97

Author(s):

J. E. Sligh ◽

S. E. Levy ◽

K. G. Waymire ◽

P. Allard ◽

D. L. Dillehay ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Germ Line ◽

Embryonic Stem ◽

Chimeric Mice ◽

Germ Line Transmission

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Epigenetic Alterations Associated with the Overall Survival and Recurrence Free Survival among Oral Squamous Cell Carcinoma Patients

Journal of Clinical Medicine ◽

10.3390/jcm9041035 ◽

2020 ◽

Vol 9 (4) ◽

pp. 1035 ◽

Cited By ~ 1

Author(s):

Yasmen Ghantous ◽

Aysar Nashef ◽

Imad Abu-Elnaaj

Keyword(s):

Overall Survival ◽

Squamous Cell Carcinoma ◽

Oral Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Clinical Features ◽

Squamous Cell ◽

Methylation Status ◽

Recurrence Free Survival ◽

Epigenetic Alterations ◽

Free Survival

Oral squamous cell carcinoma (OSCC) is a fatal disease caused by complex interactions between environmental, genomic, and epigenetic alterations. In the current study, we aimed to identify clusters of genes whose promoter methylation status correlated with various tested clinical features. Molecular datasets of genetic and methylation analysis based on whole-genome sequencing of 159 OSCC patients were obtained from the The Cancer Genome Atlas (TCGA) data portal. Genes were clustered based on their methylation status and were tested for their association with demographic, pathological, and clinical features of the patients. Overall, seven clusters of genes were revealed that showed a significant association with the overall survival/recurrence free survival of patients. The top ranked genes within cluster 4, which showed the worst prognosis, primarily acted as paraneoplastic genes, while the genes within cluster 6 primarily acted as anti-tumor genes. A significant difference was found regarding the mean age in the different clusters. No significant correlation was found between the tumor staging and the different clusters. In conclusion, our result provided a proof-of-principle for the existence of phenotypic diversity among the epigenetic clusters of OSCC and demonstrated the utility of the use epigenetics alterations in devolving new prognostic and therapeutics tools for OSCC patients.

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MRG15 Regulates Embryonic Development and Cell Proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.25.8.2924-2937.2005 ◽

2005 ◽

Vol 25 (8) ◽

pp. 2924-2937 ◽

Cited By ~ 47

Author(s):

Kaoru Tominaga ◽

Bhakti Kirtane ◽

James G. Jackson ◽

Yuji Ikeno ◽

Takayoshi Ikeda ◽

...

Keyword(s):

Cell Proliferation ◽

Embryonic Development ◽

Chromatin Remodeling ◽

Histone Deacetylases ◽

Globin Gene ◽

Immunohistochemical Analysis ◽

Wild Type ◽

Proliferating Cells ◽

Globin Promoter

ABSTRACT MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15 − / − embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15 − / − mouse embryonic fibroblasts. The hearts of the Mrg15 − / − embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15 − / − embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

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Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽

10.1242/dev.99.1.15 ◽

1987 ◽

Vol 99 (1) ◽

pp. 15-23

Author(s):

L.D. Etkin ◽

B. Pearman

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Copy Number ◽

Germ Line ◽

Gene Promoter ◽

Early Gene ◽

Mosaic Pattern ◽

Exogenous Dna ◽

Gene Coding ◽

Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

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No evidence for germ-line transmission following prenatal and early postnatal AAV-mediated gene delivery

The Journal of Gene Medicine ◽

10.1002/jgm.718 ◽

2005 ◽

Vol 7 (5) ◽

pp. 630-637 ◽

Cited By ~ 14

Author(s):

Marcus Jakob ◽

Christiane Mühle ◽

Jung Park ◽

Susi Weiß ◽

Simon Waddington ◽

...

Keyword(s):

Gene Delivery ◽

Germ Line ◽

Germ Line Transmission

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Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Biology ◽

10.3390/biology11010102 ◽

2022 ◽

Vol 11 (1) ◽

pp. 102

Author(s):

De-Li Shi

Keyword(s):

Germ Line ◽

Gene Products ◽

Transgenic Expression ◽

Guide Rna ◽

Guide Rnas ◽

Transgenic Embryos ◽

Mutant Phenotypes ◽

Biallelic Mutations ◽

Genome Activation ◽

Maternal Gene

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

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