CAREER: Analyzing and engineering multi-gene cooperation to generate cell lines for therapeutic protein production

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。[0846392]智力优势：作为细胞生物学家、遗传学家或工程师，我们通常会过度表达细胞中的某个基因，观察该细胞，并将其行为与母体进行比较。然而，决定细胞表型的往往是多个基因，而不是一个基因。一个例子是永生化哺乳动物细胞的持续增殖特性。改变单个基因表达或单个蛋白质活性的突变很少足以使细胞永生；相反，多个基因通常需要突变。本项目研究了多种基因如何影响永生化细胞的表型：增殖、凋亡、衰老和血清不依赖生长。该项目还旨在确定不同的基因表达水平如何影响这些表型。换句话说，实验涉及组合效应和剂量效应。更广泛的影响：这项技术提供了一种强大的工具，可以产生具有优化性能的细胞系，用于生产治疗性蛋白质。利用这个实验和分析平台，该项目将产生在低血清或无血清培养基中表现出高增殖率的细胞系。各种基因组合的表达将被评估以改善或创造细胞系的能力。候选基因包括已知的致癌基因、肿瘤抑制基因和细胞周期调节基因，以及先前在小鼠中通过逆转录病毒诱变确定的基因。长期目标是产生用于生产单克隆抗体的人血浆B细胞系。斯坦福大学化学工程系的教师、研究生和本科生将收养库比蒂诺高中的一年级新生。这门课参加了一个名为AVID（通过个人决定进步）的项目。该项目对课堂表现不佳、但出勤率高、纪律问题最少的学生进行甄别。该项目没有基于种族或性别的选拔标准，尽管大多数学生都是经济上处于不利地位、未被充分代表的少数民族，他们将是家庭中第一个上大学的人。这个为期四年的项目旨在帮助这些学生从高中毕业，进入大学。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).0846392WangIntellectual Merit: As cell biologists, geneticists, or engineers, one routinely over-expresses a gene in a cell, observes the cell, and compares its behavior to the parent. Yet multiple genes, not one, often determine cell phenotypes. One example is the sustained proliferation characteristic of immortalized mammalian cells. A mutation that changes the expression of a single gene or activity of a single protein rarely suffices to immortalize a cell; instead, multiple genes generally need to be mutated. This project investigates how multiple genes contribute to the phenotypes of immortalized cells: proliferation, apoptosis, senescence, and serum-independent growth. This project also aims to determine how different gene expression levels affect these phenotypes. In other words, the experiments address both combinatorial and dose effects. Broader Impact: This technology provides a powerful tool to generate cell lines with properties optimized for the production of therapeutic proteins. Using this experimental and analytical platform, this project will generate cell lines that exhibit high proliferation rates in low serum or serum-free media. Expression of various gene combinations will be evaluated for the ability to improve or create cell lines. Gene candidates include known oncogenes, tumor suppressor genes, and cell cycle regulators, as well as genes identified previously by retroviral mutagenesis in mice. The long-term goal is to generate human plasma B cell lines for the production of monoclonal antibodies.Faculty, graduate students, and undergraduates from the Department of Chemical Engineering at Stanford University will adopt a class of freshmen at Cupertino High School. This class is enrolled in a program named AVID (Advancement via Individual Determination). The program identifies students with poor class performance, but good attendance and minimal disciplinary problems. The program has no selection criteria based on race or gender, although the majority of students are economically disadvantaged, underrepresented minorities who will be the first in their families to attend college. The four-year program aims to help these students graduate from high school and go to college.