RESUMO
Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.
Assuntos
Blastocisto/metabolismo , Proteínas de Ligação a DNA/deficiência , Células Germinativas/fisiologia , Proteínas de Ligação a RNA/genética , Espermátides/metabolismo , Fatores de Transcrição/deficiência , Animais , Blastocisto/patologia , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias , Feminino , Técnicas de Inativação de Genes , Engenharia Genética , Células Germinativas/transplante , Masculino , Camundongos , Modelos Animais , Células-Tronco Pluripotentes , Ratos , Fatores de Transcrição/genética , TranscriptomaRESUMO
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.
Assuntos
Quinase 4 Dependente de Ciclina/genética , Distrofina/genética , Morfolinos/farmacologia , Distrofia Muscular de Duchenne/terapia , Telomerase/genética , Animais , Linhagem Celular , Cães , Éxons/genética , Terapia Genética , Humanos , Camundongos , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Sítios de Splice de RNA/genéticaRESUMO
Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.
Assuntos
Proteínas de Ligação a DNA/genética , Células Germinativas/citologia , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Animais , Cruzamentos Genéticos , Proteínas de Ligação a DNA/fisiologia , Feminino , Gástrula/fisiologia , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Masculino , Camundongos , Proteínas de Ligação a RNA/fisiologia , Ratos , Processos de Determinação Sexual , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies, we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition, keeping cell density low during endoderm induction is important for the efficiency. On the other hand, we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm, indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless, these results should enable better control in the production of definitive endoderm-derived cells.
