基于CRISPR/Cas9基因编辑技术的本科教学实验体系的创建与实践

CRISPR/Cas9是新兴的基因编辑技术,在生命科学研究中发挥着重要的作用。将它引入本科生的实验教学,使本科生了解这项前沿科研技术很有意义。我们创建了一个基于CRISPR/ Cas9技术的本科教学实验体系。该实验体系侧重CRISPR/Cas9技术在哺乳动物细胞中的应用,选用一株基因组上被插入mCherry基因的小鼠胚胎成纤维细胞为实验材料,命名为STO-82。首先设计靶向mCherry的sgRNA,构建CRISPR-Cas9/sgRNA共表达质粒。经测序验证无误后,转染到STO-82细胞。采用流式细胞仪分析检测mCherry阴性和阳性两群细胞,分选出阴性单细胞并扩大培养。最后用测序检验单克隆细胞中靶标DNA序列的编辑情况。结果显示,靶位点有插入或缺失突变,说明体系创建成功。该实验体系将sgRNA设计、CRISPR-Cas9/sgRNA共表达质粒的构建、细胞转染、单细胞分选、单克隆细胞培养、测序序列分析等内容融合为一个综合实验,用于高年级本科生的实验教学。根据实际情况,将教学实践内容分解分块教学,也可以做完整性项目教学。本教学实践采用10人左右的小班分块教学,2人一组,经过3个班（共13组）的实践,绝大部分学生都能完成实验,得到预期结果。通过这个实验,学生加深了对CRISPR/Cas9技术的原理和实验流程的理解,锻炼了实验操作能力和严谨的科研思维,也使学生对该技术的医疗应用风险有了一些认识。     

辽宁省三级保护植物木根黄芩考证及其与京黄芩比较形态学研究

木根黄芩(Scutellaria planipes Nakai et Kitag.)是唇形科黄芩属(Scutellaria L.)多年生草本,目前仅发现在辽宁省凌源市境内有零星分布,被辽宁省列为三级保护植物。该种发表于1934年,但《中国植物志》、Flora of China、《中国生物物种名录》均将其定为京黄芩(Scutellaria pekinensis Maxim.)的异名。为了阐明二者的差异性,本文首先对木根黄芩的原始文献、模式标本、模式产地、历史分布区及现今发现地进行考证,其次对二者进行差异比较研究,认为木根黄芩与京黄芩无论在分布范围和生境上,还是在宏观外部形态和微观花粉粒形态上均存在明显差异,确定木根黄芩为独立种,同时还对木根黄芩的果实进行形态补充描述。本文确定木根黄芩独立种的地位,对保护野生木根黄芩具有重要意义,同时对其药用价值的进一步研究提供了更为清晰的样本。     

染色质重塑调控生物温度适应性的研究进展

温度是限制物种适应性分布的重要环境因子,对极端环境温度的耐受性决定生物分布和扩散范围,而表观遗传可以提供快速的响应机制,促使生物快速适应极端环境温度。染色质重塑作为表观遗传的重要组成部分之一,其可以通过调控胁迫相关基因的表达从而促进生物适应不良环境条件。本文主要阐述了染色质重塑复合物的分类、组成和染色质重塑的方式,梳理了染色质重塑在生物温度适应性中的研究进展,提出染色质重塑在生物适应不良环境温度过程中发挥重要作用,并对未来染色质重塑与温度适应性研究提出建议。     

Is the edTPA the Right Choice for Evaluating Teacher Readiness?

The purpose of this article is to describe and analyze the edTPA, a performance assessment created by the Stanford Center for Assessment, Learning, and Equity (SCALE) and administered by Pearson, Inc., to assess the professional readiness of student teachers. We challenge claims made in support of using this assessment, specifically within the context of arts teacher preparation programs, and we address areas of immediate critical concern to make alternative recommendations.     

Do You Really Want to Know? Elementary Music Personnel and Potential in Utah

This is the second of two articles reporting the results of a study by the author regarding the status of elementary music education in the state of Utah. This article focuses on the qualifications of Utah's elementary music teachers (music certified, elementary classroom certified, artists-in-residence, volunteers, and paraprofessionals) and the conditions under which they teach. Interactions among teacher qualifications and teaching status are explored. Paraprofessionals play a significant role in Utah's elementary music programs. While over 90 percent of elementary schools in the United States provide regular music instruction taught by certified music specialists, less than 10 percent of Utah's elementary population receives such instruction. Nearly half of the elementary students in the state receive no designated music instruction beyond that provided by their regular elementary classroom teacher. The influence of school funding, No Child Left Behind and other accountability measures, high-stakes testing, urban/rural populations, and leadership are highlighted. Policy considerations are discussed.     

Efficient rescue of threatened biodiversity data using reBiND workflows

Abstract

Biodiversity data generated in the context of research projects often lack a strategy for long-term preservation and availability, and are therefore at risk of becoming outdated and finally lost. The reBiND project aims to develop an efficient and well-documented workflow for rescuing such data sets. The workflow consists of phases for data transformation into contemporary standards, data validation, storage in a native XML database, and data publishing in international biodiversity networks. It has been developed and tested using the example of collection and observational data but is flexible enough to be transferred to other data types and domains.     

ABCG2/BCRP Dysfunction as a Major Cause of Gout

Recent genome-wide association studies showed that serum uric acid (SUA) levels relate to ABCG2/BCRP gene, which locates in a gout-susceptibility locus revealed by a genome-wide linkage study. Together with the ABCG2 characteristics, we hypothesized that ABCG2 transports urate and its dysfunction causes hyperuricemia and gout. Transport assays showed ATP-dependent transport of urate via ABCG2. Kinetic analysis revealed that ABCG2 mediates high-capacity transport of urate (Km: 8.24 ± 1.44 mM) even under high-urate conditions. Mutation analysis of ABCG2 in 90 Japanese hyperuricemia patients detected six nonsynonymous mutations, including five dysfunctional variants. Two relatively frequent dysfunctional variants, Q126X and Q141K, were then examined. Quantitative trait locus analysis of 739 Japanese individuals showed that Q141K increased SUA as the number of minor alleles of Q141K increased (p = 6.60 × 10^?5). Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. Becuase Q126X and Q141K are assigned to nonfunctional and half-functional haplotypes, respectively, their genotype combinations are divided into four functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those with dysfunctional ABCG2 increased the gout risk, especially those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10^?21). These genotypes were found in 10.1% of gout patients, but in only 0.9% of control. Our function-based clinicogenetic (FBCG) analysis showed that combinations of the two dysfunctional variants are major causes of gout, thereby providing a new approach for prevention and treatment of the gout high-risk population.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

THE LAKE ST. LUCIA SYSTEM—A RESEARCH ASSESSMENT

SUMMARY

This paper assesses the research undertaken at Lake St. Lucia over the past 25 years based on over 300 documents from that period. Trends related to both time and subject matter are evident, and these are considered in relation to the gaps in our current knowledge concerning the system.

A feature evident throughout the period under consideration is that the major portion of documented material available relates to reports and contributions to workshops (77%) with only 23% from scientific publications. Contributions by these two sources to the subject group being considered for St. Lucia are markedly different with research publications dominating the biological field and reports dominating in physical aspects, catchment characteristics, man's activities, management, dredging and hydrological modelling. However, some 55% of all unpublished data related to reviews or assessments of the state of research on St. Lucia.

The importance of the scientific publications group as an indicator of the state of research into the system is considered in the light of an apparent decline in the number of completed projects being published. It is also considered in the light of the recent establishment of a co-ordinated Lake St. Lucia Research Programme, which may provide the impetus for a more concentrated and directed research effort on the Lake System.