RESUMO
CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Embrião de Mamíferos/metabolismo , Endonucleases/metabolismo , Edição de Genes , Marcação de Genes/métodos , RNA Guia de Cinetoplastídeos/metabolismo , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Edição de Genes , Marcação de Genes , Engenharia Genética , Camundongos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , ZigotoRESUMO
BACKGROUND: Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. METHODS: We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. RESULTS: We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. CONCLUSION: This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.