RESUMO
The recent success of derivation of mammalian haploid embryonic stem cells (haESCs) has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons. Using piggyBac transposon-based insertional mutagenesis, we generated a haploid neural cell library harboring genome-wide mutations for genetic screening. As a proof of concept, we screened for Mn2+-mediated toxicity and identified the Park2 gene. Our findings expand the applications of mouse haploid cell technology to somatic cell types and may also shed light on the mechanisms of ploidy maintenance.
Assuntos
Testes Genéticos , Genoma , Haploidia , Amidas/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diploide , Camundongos , Mitose/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pâncreas/citologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Pluripotent stem cells can be evaluated by pluripotent markers expression, embryoid body aggregation, teratoma formation, chimera contribution and even more, tetraploid complementation. Whether iPS cells in general are functionally equivalent to normal ESCs is difficult to establish. Here, we present the detailed procedure for chimera formation and tetraploid complementation, the most stringent criterion, to assessing pluripotency.
