11 research outputs found

    Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing

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    <div><p>The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.</p></div

    The sgRNA:Cas9-mediated modifications in Cas9 expression oocytes.

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    <p>(A and E) Schematic of two sgRNAs targeting the mouse <i>Ar</i> gene and NLRP3 gene. (B) PCR products of the targeted, and detection of Cas9-mediated on-target cleavage on wild-type zygotes by co-injection Cas9 mRNA and Ar sgRNAs by T7EI cleavage assay. (C and F) PCR products of the targeted, and detection of Cas9-mediated on-target cleavage on Zp3-Cas9 transgenic mouse zygotes injected with sgRNA only by T7EI cleavage assay. (D and G) Sequencing results of modified alleles in Zp3-Cas9 transgenic mouse zygotes injected with sgRNAs.</p

    The sgRNA: Cas9-mediated off-target cleavage <i>in vivo</i>.

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    <p>(A) PCR products of the targeted, and detection of Cas9-mediated AR-A OTS1 off-target cleavage by T7EI cleavage assay. Lower panel, sequencing results of mutant off-target sites. (B) PCR products of the targeted, and detection of Cas9-mediated AR-B OTS9 off-target cleavage by T7EI cleavage assay. Lower panel, sequencing results of mutant off-target sites. (C to E) PCR products of the targeted, and detection of Cas9-mediated off-target cleavage by T7EI cleavage assay on OTS2, OTS3 and OTS11.</p

    Generating a oocyte-specific Cas9 transgenic mouse line.

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    <p>(A) A schematic representation of the vector expressing Cas9 in oocyte. The Zp3-Cas9 plasmid encodes the Cas9 under the regulation of a 2 kb Zp3 promoter. Two nuclear localization signals (NLSs) were inserted into N and C terminals of Cas9. (B) Six transgenic founders from total 39 F0 were identified by Cas9 gene amplification by PCR. PC, positive control. (#21, #22, #23 are germ-line heritable). (C and D) RT-PCR and Western blotting confirmed the ovary specific expression of Cas9 in Zp3-Cas9 transgenic mouse.</p

    Image_5_Global Burden of cardiomyopathy and myocarditis in the older adults from 1990 to 2019.TIF

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    BackgroundCardiomyopathy and myocarditis (CM-MC) are common chronic diseases causing heart failure in older adults. We aimed to analyze the burden of CM-MC in older adults aged 60–89 years at the global, regional, and national levels in 204 countries from 1990 to 2019.MethodsDetailed data on CM-MC from 1990 to 2019 were analyzed from the Global Burden of Diseases Study 2019, including incidence, mortality, disability-adjusted life years (DALYs) and the proportion of deaths caused by different risks factors. All results are presented as numbers, age-standardized rates per 100,000 person-years and estimated annual percentage change (EAPC) with an uncertainty interval of 95%.ResultsGlobally, there were 475,458 (339,942–638,363) incidence cases from CM-MC in 2019; with an age-standardized incidence rate (ASIR) of 16 (13–19.3) per 100,000 person-years. And there were 185,308 (154,610–200,448) deaths, with the age-standardized mortality rate (ASMR) being 4.4 (3.7–4.8). CM-MC resulted in 3,372,716 (2,931,247–3,693,622) DALYs, with an age-standardized DALYs rate (ASDR) of 114.8 (98.7–126.1). Estimated annual percentage change (EAPCs) for ARIS, ARMS, and ARDS has decreased. At the national level, the United States of America had the highest mortality [21,372 (18,924–24,241)] and disability-adjusted life years [407,712 (370,234–470,165)]. And China had the highest number of incident cases [122, 266 (85,925–166,095)]. Globally, high systolic blood pressure and alcohol consumption were the top two risk factors for the proportion of CM-MC deaths.ConclusionCM-MC is still an important cause of early death and chronic disability in older adults. Based on this study, public health agencies should seek more effective methods to prevent and treat CM-MC.</p

    Image_1_Global Burden of cardiomyopathy and myocarditis in the older adults from 1990 to 2019.TIF

    No full text
    BackgroundCardiomyopathy and myocarditis (CM-MC) are common chronic diseases causing heart failure in older adults. We aimed to analyze the burden of CM-MC in older adults aged 60–89 years at the global, regional, and national levels in 204 countries from 1990 to 2019.MethodsDetailed data on CM-MC from 1990 to 2019 were analyzed from the Global Burden of Diseases Study 2019, including incidence, mortality, disability-adjusted life years (DALYs) and the proportion of deaths caused by different risks factors. All results are presented as numbers, age-standardized rates per 100,000 person-years and estimated annual percentage change (EAPC) with an uncertainty interval of 95%.ResultsGlobally, there were 475,458 (339,942–638,363) incidence cases from CM-MC in 2019; with an age-standardized incidence rate (ASIR) of 16 (13–19.3) per 100,000 person-years. And there were 185,308 (154,610–200,448) deaths, with the age-standardized mortality rate (ASMR) being 4.4 (3.7–4.8). CM-MC resulted in 3,372,716 (2,931,247–3,693,622) DALYs, with an age-standardized DALYs rate (ASDR) of 114.8 (98.7–126.1). Estimated annual percentage change (EAPCs) for ARIS, ARMS, and ARDS has decreased. At the national level, the United States of America had the highest mortality [21,372 (18,924–24,241)] and disability-adjusted life years [407,712 (370,234–470,165)]. And China had the highest number of incident cases [122, 266 (85,925–166,095)]. Globally, high systolic blood pressure and alcohol consumption were the top two risk factors for the proportion of CM-MC deaths.ConclusionCM-MC is still an important cause of early death and chronic disability in older adults. Based on this study, public health agencies should seek more effective methods to prevent and treat CM-MC.</p

    Image_3_Global Burden of cardiomyopathy and myocarditis in the older adults from 1990 to 2019.TIF

    No full text
    BackgroundCardiomyopathy and myocarditis (CM-MC) are common chronic diseases causing heart failure in older adults. We aimed to analyze the burden of CM-MC in older adults aged 60–89 years at the global, regional, and national levels in 204 countries from 1990 to 2019.MethodsDetailed data on CM-MC from 1990 to 2019 were analyzed from the Global Burden of Diseases Study 2019, including incidence, mortality, disability-adjusted life years (DALYs) and the proportion of deaths caused by different risks factors. All results are presented as numbers, age-standardized rates per 100,000 person-years and estimated annual percentage change (EAPC) with an uncertainty interval of 95%.ResultsGlobally, there were 475,458 (339,942–638,363) incidence cases from CM-MC in 2019; with an age-standardized incidence rate (ASIR) of 16 (13–19.3) per 100,000 person-years. And there were 185,308 (154,610–200,448) deaths, with the age-standardized mortality rate (ASMR) being 4.4 (3.7–4.8). CM-MC resulted in 3,372,716 (2,931,247–3,693,622) DALYs, with an age-standardized DALYs rate (ASDR) of 114.8 (98.7–126.1). Estimated annual percentage change (EAPCs) for ARIS, ARMS, and ARDS has decreased. At the national level, the United States of America had the highest mortality [21,372 (18,924–24,241)] and disability-adjusted life years [407,712 (370,234–470,165)]. And China had the highest number of incident cases [122, 266 (85,925–166,095)]. Globally, high systolic blood pressure and alcohol consumption were the top two risk factors for the proportion of CM-MC deaths.ConclusionCM-MC is still an important cause of early death and chronic disability in older adults. Based on this study, public health agencies should seek more effective methods to prevent and treat CM-MC.</p

    Image_2_Global Burden of cardiomyopathy and myocarditis in the older adults from 1990 to 2019.TIF

    No full text
    BackgroundCardiomyopathy and myocarditis (CM-MC) are common chronic diseases causing heart failure in older adults. We aimed to analyze the burden of CM-MC in older adults aged 60–89 years at the global, regional, and national levels in 204 countries from 1990 to 2019.MethodsDetailed data on CM-MC from 1990 to 2019 were analyzed from the Global Burden of Diseases Study 2019, including incidence, mortality, disability-adjusted life years (DALYs) and the proportion of deaths caused by different risks factors. All results are presented as numbers, age-standardized rates per 100,000 person-years and estimated annual percentage change (EAPC) with an uncertainty interval of 95%.ResultsGlobally, there were 475,458 (339,942–638,363) incidence cases from CM-MC in 2019; with an age-standardized incidence rate (ASIR) of 16 (13–19.3) per 100,000 person-years. And there were 185,308 (154,610–200,448) deaths, with the age-standardized mortality rate (ASMR) being 4.4 (3.7–4.8). CM-MC resulted in 3,372,716 (2,931,247–3,693,622) DALYs, with an age-standardized DALYs rate (ASDR) of 114.8 (98.7–126.1). Estimated annual percentage change (EAPCs) for ARIS, ARMS, and ARDS has decreased. At the national level, the United States of America had the highest mortality [21,372 (18,924–24,241)] and disability-adjusted life years [407,712 (370,234–470,165)]. And China had the highest number of incident cases [122, 266 (85,925–166,095)]. Globally, high systolic blood pressure and alcohol consumption were the top two risk factors for the proportion of CM-MC deaths.ConclusionCM-MC is still an important cause of early death and chronic disability in older adults. Based on this study, public health agencies should seek more effective methods to prevent and treat CM-MC.</p

    Image_4_Global Burden of cardiomyopathy and myocarditis in the older adults from 1990 to 2019.TIF

    No full text
    BackgroundCardiomyopathy and myocarditis (CM-MC) are common chronic diseases causing heart failure in older adults. We aimed to analyze the burden of CM-MC in older adults aged 60–89 years at the global, regional, and national levels in 204 countries from 1990 to 2019.MethodsDetailed data on CM-MC from 1990 to 2019 were analyzed from the Global Burden of Diseases Study 2019, including incidence, mortality, disability-adjusted life years (DALYs) and the proportion of deaths caused by different risks factors. All results are presented as numbers, age-standardized rates per 100,000 person-years and estimated annual percentage change (EAPC) with an uncertainty interval of 95%.ResultsGlobally, there were 475,458 (339,942–638,363) incidence cases from CM-MC in 2019; with an age-standardized incidence rate (ASIR) of 16 (13–19.3) per 100,000 person-years. And there were 185,308 (154,610–200,448) deaths, with the age-standardized mortality rate (ASMR) being 4.4 (3.7–4.8). CM-MC resulted in 3,372,716 (2,931,247–3,693,622) DALYs, with an age-standardized DALYs rate (ASDR) of 114.8 (98.7–126.1). Estimated annual percentage change (EAPCs) for ARIS, ARMS, and ARDS has decreased. At the national level, the United States of America had the highest mortality [21,372 (18,924–24,241)] and disability-adjusted life years [407,712 (370,234–470,165)]. And China had the highest number of incident cases [122, 266 (85,925–166,095)]. Globally, high systolic blood pressure and alcohol consumption were the top two risk factors for the proportion of CM-MC deaths.ConclusionCM-MC is still an important cause of early death and chronic disability in older adults. Based on this study, public health agencies should seek more effective methods to prevent and treat CM-MC.</p
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