Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment ...strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is ...tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro.
Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA ...content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for understanding the recurrence risks for mtDNA diseases.
Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, ...clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation.
X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In ...contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.
•Cellular senescence phenotypes are triggered by hormone ablation in an animal level.•Secretory phenotypes and anti-oxidant responses were accompanied in human specimens.•These responses are novel ...targets for prevention of endocrine resistance.
Endocrine resistance is a major problem in prostate cancer. Recent studies suggest that cellular plasticity plays a key role in therapy resistance. Yet little is known about the cellular changes of human prostate cancer after androgen deprivation therapy (ADT). In this study, we investigated cellular senescence, senescence-associated secretory phenotypes (SASPs), and anti-oxidant responses. Hormone ablation upregulated senescence-associated (SA)-β-Gal activity in prostate glands, as well as the expressions of p27KIP1 and p53, in a mouse castration model. In line with this, the expressions of p21CIP1 and p27KIP1 were significantly more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. In a study of SASP markers, the expressions of IL6 and IL8 were also more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. IL6, IL8, and MMP2 were expressed more strongly in human prostate cancer specimens resected after ADT than in untreated tumors. Of note, treatment with the anti-oxidant reagent NAC significantly suppressed SA-β-Gal activity in androgen-sensitive human prostate cancer LNCaP cells. In immunohistochemical analyses on anti-oxidant response genes, NRF2 and NQO1 were more upregulated after hormone ablation in human prostate gland and carcinoma specimens after ADT than in untreated specimens or in murine prostate glands after castration. Taken together, these findings suggest that ADT induces cellular senescence processes accompanied by secretory phenotypes and anti-oxidant responses in prostate. These cellular changes may be attractive targets for preventing endocrine resistance in prostate cancer.
In mammals, complementary contributions of both the maternal and the paternal genomes are required for normal development because of the parental-allele-specific modification of the genome, called ...genomic imprinting. Therefore, parthenogenetic embryos (PG) with two maternal genomes cannot develop to term, and PG chimeras show a restricted cell contribution of donor cells and reduced weight, although they can develop to term. On the other hand, parthenogenetic embryonic stem cells (PGES) chimeras are more normal in their tissue contribution of donor cells and body weight compared with PG chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the imprint status in donor cells of PGES and PG chimeras. In somatic lineages, genomic imprinting was lost in some PGES chimeras, whereas those in PG chimeras were almost totally maintained. Moreover, loss of imprints correlated to the gene expression pattern of imprinted genes. Therefore, this loss of imprinting in PGES chimeras could improve the tissue contribution and body weight to a normal level. On the other hand, in germ lineages, both PGES and PG in chimeras showed normal erasure of imprints, indicating that the reprogramming in germ lineages is an inevitable event, regardless of the imprint status of primordial germ cells.
Mitochondria play a crucial role in the development and function of germ cells. Mitochondria contain a maternally inherited genome that should be transmitted to offspring without reactive oxygen ...species-induced damage during germ line development. Germ cells are also involved in the mitochondrial DNA (mtDNA) bottleneck; thus, the appropriate regulation of mtDNA in these cells is very important for this characteristic transmission. In this review, we focused on unique regulation of the mitochondrial genome in animal germ cells; paternal elimination and the mtDNA bottleneck in females. We also summarized the mitochondrial nucleoid factors involved in various mtDNA regulation pathways. Among them, mitochondrial transcription factor A (TFAM), which has pleiotropic and essential roles in mtDNA maintenance, appears to have putative roles in germ cell regulation.
We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized ...oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.
Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of ...function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca2+/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.
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