CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate

Environmental factors, epigenetics, and developmental origin of reproductive disorders

Sex-specific differentiation, development, and function of the reproductive system are largely dependent on steroid hormones. For this reason, developmental exposure to estrogenic and anti-androgenic endocrine disrupting chemicals (EDCs) is associated with reproductive dysfunction in adulthood. Human data in support of “Developmental Origins of Health and Disease” (DOHaD) comes from multigenerational studies on offspring of diethylstilbestrol-exposed mothers/grandmothers. Animal data indicate that ovarian reserve, female cycling, adult uterine abnormalities, sperm quality, prostate disease, and mating behavior are susceptible to DOHaD effects induced by EDCs such as bisphenol A, genistein, diethylstilbestrol, p,p′-dichlorodiphenyl-dichloroethylene, phthalates, and polyaromatic hydrocarbons. Mechanisms underlying these EDC effects include direct mimicry of sex steroids or morphogens and interference with epigenomic sculpting during cell and tissue differentiation. Exposure to EDCs is associated with abnormal DNA methylation and other epigenetic modifications, as well as altered expression of genes important for development and function of reproductive tissues. Here we review the literature exploring the connections between developmental exposure to EDCs and adult reproductive dysfunction, and the mechanisms underlying these effects

Introducing trainees to research using an online, asynchronous course

Abstract Introduction: Research is an important aspect of many students’ training. However, formal research training is rarely included in curricula. Thus, we developed an online, asynchronous series of modules to introduce trainees to multiple topics that are relevant to the conduct of research. Methods: Research 101 was utilized by first-year medical students and undergraduate students conducting mentored research projects. Students’ knowledge, confidence, and satisfaction were assessed using pre- and post-module surveys with five-point Likert scaled questions, open-ended text responses, and a final quiz. Results: Pre-module survey results showed that learners felt most confident with the Conducting a literature search and Race and racism in medicine modules and least confident with the Submitting an Institutional Review Board protocol at UC module. Post-module survey responses were significantly increased compared to pre-module results for all modules and questions (p < 0.0001). The response to “The content of this module met my needs” was endorsed across all modules (84.9% “yes” responses). A final quiz of 25 multiple-choice questions was completed by 92 participants who received a median score of 21. Content analysis of open-ended post-module survey responses identified several strengths and opportunities for improvement in course content and instructional methods. Conclusions: These data demonstrate that significant learning resulted from completion of Research 101, as post-module survey scores were significantly higher than pre-module survey scores for all modules and questions. Final quiz scores were positive but also highlighted opportunity for additional trainee learning and will guide evolution of future modules

Linker of nucleoskeleton and cytoskeleton complex proteins in cardiac structure, function, and disease

The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, composed of proteins within the inner and the outer nuclear membranes, connects the nuclear lamina to the cytoskeleton. The importance of this complex has been highlighted by the discovery of mutations in genes encoding LINC complex proteins, which are causative for skeletal or cardiac myopathies. Herein, this review summarizes structure, function, and interactions of major components of the LINC complex, highlights how mutations in these proteins may lead to cardiac disease, and outlines future challenges in the field

Barth Syndrome Cardiomyopathy: An Update

Barth syndrome (BTHS) is an X-linked mitochondrial lipid disorder caused by mutations in the TAFAZZIN (TAZ) gene, which encodes a mitochondrial acyltransferase/transacylase required for cardiolipin (CL) biosynthesis. Cardiomyopathy is a major clinical feature of BTHS. During the past four decades, we have witnessed many landmark discoveries that have led to a greater understanding of clinical features of BTHS cardiomyopathy and their molecular basis, as well as the therapeutic targets for this disease. Recently published Taz knockout mouse models provide useful experimental models for studying BTHS cardiomyopathy and testing potential therapeutic approaches. This review aims to summarize key findings of the clinical features, molecular mechanisms, and potential therapeutic approaches for BTHS cardiomyopathy, with particular emphasis on the most recent studies

CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate