Molecular and Cellular Biology Techniques

核心D提供技术专业知识和最先进的分子和细胞生物学方法 项目中所描述的蛋白质的克隆和表达。这种集中操作协调了 所有分子生物学都发挥作用，并确保所有蛋白质以统一的方式被克隆和表达 时尚。这一点尤其重要，因为几名调查人员计划分析一些 同样的蛋白质。这种一致性大大提高了蛋白质之间比较的准确性。 应以下要求设计和克隆编码野生型或突变蛋白的表达载体 个人调查员。该计划项目中描述的所有野生型基因都被鉴定、测序和 组装在克隆载体中。因此，表达野生型或突变蛋白的载体可以从 CDNA。表达共有序列多肽的载体将从人工合成的 寡核苷酸。取决于对蛋白质分析的要求(量和/或修改 蛋白质)，这些表达载体被设计用于在原核或真核细胞系中表达。核心D 还建立表达蛋白质的细胞系，确定最佳蛋白质表达的条件，并 艾滋病病毒在表达蛋白的纯化中。 因此，核心D的服务包括：a)对最佳表达系统的建议，b)设计 表达所需蛋白质的方法，c)通过限制性内切酶和/或DNA确认野生型DNA序列 序列分析，d)设计用于DNA序列分析的引物，e)设计用于 野生型或突变型构建体，f)提取DNA，g)用限制性内切酶消化DNA，h)通过 聚合酶链式反应，i)在克隆和表达载体中连接DNA(片段)，j)将连接的DNA转化/转染到细胞中 表达蛋白质的细胞系，k)选择克隆，i)扩增克隆，m)分离永久转染的细胞系， N)保持转化的细胞系，o)建立最佳的蛋白质表达条件，p)辅助蛋白质 纯化，q)保持携带野生型和表达载体的细胞系。 Core D的所有人员都在培训与分子生物学和细胞培养项目相关的新用户 技术，并监督细胞培养设施的正确使用。这确保了电池系统的安全维护 并保证遵守NIH的指导方针。 核心D设计和克隆编码蛋白质的表达载体 个别项目。他们建立细胞系，确定蛋白质表达的最佳条件，并帮助 在表达蛋白的纯化过程中。

Core D provides technical expertise and state of the art molecular and cellular biology methodology for cloning and expression of the proteins described in the program project. This centralized operation coordinates all molecular biology work and ensures that all proteins are cloned and expressed in a uniform fashion. This is particularly important since several investigators plan to analyze different aspects of some of the same proteins. This uniformity greatly enhances accuracy of comparison between the proteins. Core D designs and clones expression vectors encoding wild type or mutated proteins as requested by the individual investigators. All wild type genes described in this program project are identified, sequenced and assembled in cloning vectors. Thus, vectors expressing wild type or mutated proteins can be cloned from cDNAs. Vectors expressing consensus sequence peptides will be constructed from synthetic oligonucleotides. Depending on the requirement for the analysis of the protein (amount and/or modification of protein), the expression vectors are designed for expression in prokaryotic or eukaryotic cell lines. Core D also establishes cell lines expressing the proteins, determines conditions for optimum protein expression and aids in the purification of the expressed protein. Thus the service of Core D includes: a) advice toward an optimal expression system, b) design methodologies to express desired proteins, c) confirm wild type DNA sequence by restriction and/or DNA sequence analysis, d) design primers for DNA sequence analysis, e) design primers for PCR amplification of wild type or mutant constructs, f) extract DNA, g) digest DNA with restriction enzymes, h) amplify DNA by PCR, i) ligate DNA (fragments) in cloning and expression vectors, j) transform/transfect ligated DNA into cell lines to express the proteins, k) select clones, I) amplify clones, m) isolate permanently transfected cell lines, n) maintain transformed cell lines, o) establish optimum protein expression conditions, p) assist in protein purification, q) maintain cell lines carrying wild type and expression vectors. All personnel in Core D trains new users associated with the projects in molecular biology and cell culture techniques and oversees proper use of cell culture facilities. This ensures safe maintenance of cell systems and guarantees compliance with NIH guidelines. Core D designs and clones expression vectors encoding proteins requested by the principal investigators of the individual projects. They establish cell lines, determine optimum conditions for protein expression and aid in the purification of the expressed protein.