【CASK EFFECT】0910F阅读全方位锻炼--越障【SCI】 1-9

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【CASK EFFECT】0910G阅读能力基础自测（速度、难度、深度、越障、真题、RAM）
https://bbs.gter.net/forum.php?mod=viewthread&tid=910464&highlight

【CASK EFFECT】0910F阅读全方位锻炼--越障【SCI】汇总贴
https://bbs.gter.net/thread-982020-1-1.html

规则：0 u, r. g$ C/ d+ [4 f5 C

我每天贴出1000字左右的一篇文字7 j) N0 Q, Q- ]( V4 E

没有别的要求，只要大家坚持读完就可以

如果你能坚持一个月，你会发现自己的阅读进化了~
[注]9 K7 C8 w4 {" L
1、直接在电脑屏幕面前做，虽然GRE阅读是在纸上考，但是这个过程会遏制你做笔记，同时给你的阅读造成视觉障碍，也就是把难度训练和抗干扰训练同步结合，增加效率（初期会很累，但是既然大家想要成为高手，那么就别对自己太温柔）

Biotechnology and the chicken B cell line DT40

Abstract.
Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin(Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information ( random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest with in living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection.
Copyright © 2007 S. Karger AG, Basel

Protein optimization by rational design
The increased sophistication of recombinant DNA techniques has made it possible to artificially generate mutant genes and express these in vitro in prokaryotic or eukaryotic cells and in transgenic animals. From the very beginning, this new discipline of protein engineering intended to produce variants of existing proteins to benefit human health and to improve quality of life. However, this goal often proved to be elusive due to the difficulty of predicting the structure-function relationship of engineered variants even for well characterized proteins (Pabo,1983). Attempts of intelligent protein design are therefore often limited to residuesfor which natural variation exists between homologues or which were known toplay a central role in activity. Tremendous interdisciplinary collaborations toimprove computer modeling- and rational design-based structure functionpredictions are currently under way (i.e. Rosetta, University of Washington,USA). In addition, various biotech companies have successfully specialized indeveloping and providing algorithms and computer programs for rational proteinand drug design (i.e. Modular Genetics, USA and Xencor, USA). Despite rapidprogress in the development of increasingly sophisticated rational designprograms, it is unlikely that rational design by itself will be sufficient tosolve all future demands for protein evolution and optimization (Dahiyat etal., 1997; De Maeyer et al., 2000; Klepeis et al., 2003). Indeed, the latesttrend is to use a combinatorial approach to include mutagenesis approaches in additionto in silico techniques (Bornscheuer et al., 2001; Klepeis et al., 2004).


Protein optimization by random mutagenesis
Alternatively, directed molecular evolution or evolutionary biotechnology is based on processes that mimic natural evolution to create bio-molecules with desired properties. Thus protein diversification is achieved by random mutagenesis without prior knowledge of the structure-function relation. The degree of variation within a protein population can be enhanced artificially during amplification through the use of mutator strains, error-prone DNA polymerases, base analogs or base modifying reagents (Farinas et al., 2001;Kurtzman et al., 2001; Stromgaard et al., 2004). In particular, the usage of partially randomized oligonucleotides makes it possible to achieve extremely high levels of variation. This technique has the advantage that mutations are specifically introduced at the sites of interest whereas other regions (or domains) are not affected by mutagenesis (Kunkel, 1985; Jestin et al., 2004;Hart et al., 2006). However the oligonucleotide-based technique requires detailed information of the protein structure-function relation of the particular domain, which is currently available only in very few cases.

In vitro random mutagenesis techniques have been successfully combined with the expression of proteins in phages or bacteria to generate and search for protein variants with desirable properties (Dower et al., 2002; Graddis etal., 2002). In particular, phage display systems using fusions of randomized peptides to coat proteins of filamentous phages are a very popular display system (Smith, 1985; Winter et al., 1994; O’Neil et al., 1995; Amstutz et al.,2001; Willats, 2002). Phage particles can be separated by their affinity to a given target (bio-panning). However these phage display libraries generally fail to have an intrinsic diversification platform to improve their binding properties step wise by mutation and selection. In addition, proteins improved in bacteria frequently fail to display their selected-for characteristics when expressed in vertebrate cells because protein processing differs between prokaryotic and eukaryotic cells. Therefore, the usage of a eukaryotic diversification system has a decided advantage (i.e. post-translational modifications).

An intrinsic protein diversification system in B lymphocytes
The immune system of vertebrates uses hypermutation of Ig genes and B cell selection for affinity maturation of antibodies (McKean et al.,1984; French et al., 1989; Rajewsky, 1996). Somatic hypermutation in the antibody coding immunoglobulin genes has been successfully used for antibody diversification and optimization for millions of years.

During somatic hypermutation, point mutations are introduced at a million-fold increased rate in a defined region of the immunoglobulin genes of B lymphocytes. These mutations contribute to the diversification of the antibody repertoire generated by B lymphocytes and are essential for an efficient immunoresponse to a given pathogen or microorganism (Rajewsky, 1996).

The Ig-locus specific hypermutation activity is preserved in some B cell lines (Wabl et al., 1885; Green et al., 1995; Zhang et al., 2001;Arakawa et al., 2004). Alternatively the ectopic expression of the proteinactivation-induced cytidine deaminase (AID) causes somatic hypermutation and class switch recombination in various eukaryotic non-B cell lines and even prokaryotic cells (Martin et al., 2002; Petersen- Mahrt et al., 2002).

Arakawa et al. (2004) have shown that the B cell line DT40,which normally diversifies its Ig genes by gene conversion, can be induced into Ig gene specific hypermutation (in-cell locus specific random mutagenesis). It was proposed that transgenes integrated into the Ig locus of DT40 will be specifically and efficiently diversified by hypermutation and gene conversion (depending on the presence or absence of gene conversion donor sequences). During gene conversion,DNA sequences sharing a high degree of sequence similarity to the target transgene are utilized as donors to randomly shuffle complex genetic information.

The DT40 cell line
The chicken B lymphocyte cell line DT40, transformed by an avianl eukosis virus, displays some unique properties, which make this cell line valuable for academic research and potentially also for commercial application.Buerstedde, Takeda and others observed that DT40 cells exhibit a very high fre quency of targeted-locus specific DNA integration at almost all its endogenous geneloci (Buerstedde et al., 1991; Sonoda et al., 2001). Targeting efficiencies inDT40 cells of up to 80% have been described, enabling fast and efficient genedisruption (knock out) or site specific transgene integration (knock in). This feature appears to be common among various chicken B lymphocyte cell lines,yet, it can not be seen in chicken non-B cell lines (Yamazoe et al., 2004).Presumably an overall enhanced homologous recombination activity is responsible for the remarkably increased targeting efficiency. Despite its high homologous recombinationand gene targeting efficiency, the karyotype and phenotype of DT40 cells stay quite stable even during long periods in cell culture. Therefore, successive rounds of gene disruption on multiple genes can be performed. In addition, DT40cells can easily be maintained in cell culture, have a short generation time of approximately ten hours and can be effortlessly subcloned (Yamazoe et al.,2004). Due to these features, DT40 is widely used as a cellular model system for reverse genetic studies (Sonoda et al., 2001).

cjluUID: 2448066

好长~又是生物……

紫薇花开UID: 2461167

我天天越障了  老师逼得

dairymanUID: 2683905

这个似乎是讲xxB细胞比较不错，很有研究价值

lghscuUID: 2552043

这篇读第一遍还是有点难

那就来一个第二遍吧，呵呵

bobo19890313UID: 2622130

今天晚上再读一遍，我就不信了。。。

solosandraUID: 2889127

看下来了,但是基本上完全没有看懂..
回头再看一遍..><