轉(zhuǎn)基因技術

內(nèi)容概要

　　在后基因組時代，學界所面臨的主要挑戰(zhàn)之一是如何破解大量的編碼蛋白質(zhì)的基因功能。在《轉(zhuǎn)基因技術(原理與實驗方案原著第3版導讀版)(精)》(作者卡特萊特)中，該領域的專家在第2版的基礎上進行了更新和補充，以求能夠詳盡地反映當前基因修飾技術的最新進展?！掇D(zhuǎn)基因技術(原理與實驗方案原著第3版導讀版)(精)》不僅包括基因修飾小鼠制作過程，同時也介紹了其他模式生物的轉(zhuǎn)基因技術，以及在顯微注射、位點特異性重組系統(tǒng)、冷凍保存等方面的探索和嘗試。本書秉承Springer《分子生物學方法》系列叢書的一貫風格，闡述明晰、便于使用，每章包括對相關問題的介紹，所需材料和試劑的清單，實驗操作的具體步驟，以及常見問題的解決方法和缺陷規(guī)避。

書籍目錄

前言
撰稿人
第一部分 多種模式動物的轉(zhuǎn)基因技術
1 轉(zhuǎn)基因果蠅
2 轉(zhuǎn)基因線蟲
3 利用Tol2轉(zhuǎn)座子系統(tǒng)制作轉(zhuǎn)基因斑馬魚
4 轉(zhuǎn)基因爪蟾
5 利用DNA前核注射制作轉(zhuǎn)基因大鼠
第二部分 小鼠的轉(zhuǎn)基因技術
6 細胞類型特異的轉(zhuǎn)基因小鼠
7 設計轉(zhuǎn)基因載體和DNA轉(zhuǎn)入基因組一成功的關鍵
8 過表達轉(zhuǎn)基因一前核顯微注射
9 基因打靶載體
10 基因捕獲：基因敲除的快速通道
11 小鼠胚胎干細胞的培養(yǎng)
12 打靶胚胎干細胞
13 通過顯微注射獲得嵌合體小鼠
14 通過桑葚胚細胞聚集獲得嵌合體小鼠
15 獲得突變體小鼠相關的手術操作
16 用于小鼠基因組修飾相關的位點特異性重組酶
17 Cre轉(zhuǎn)基因小鼠
18 大規(guī)模小鼠基因突變
19 小鼠的繁殖和飼養(yǎng)
20 維持和保存實驗室變異小鼠的生物學方法
Ⅰ：保存突變小鼠株
21 維持和保存實驗室變異小鼠的生物學方法
Ⅱ：復蘇突變小鼠株
索引

章節(jié)摘錄

　　Summary　　Transgenesis in Drosophila melanogaster relies upon direct microinjection of embryos and subsequentcrossing of surviving adults．The necessity of crossing single flies to screen for transgenic events limits therange of useful transgenesis techniques to thosc that have a very high frequency of integration，So thatabout 1 in 10 to 1 in 100 surviving adult flies carry a transgene．Until recently，only random P—elementtransgenesis fulfilled these criteria．However，recent advances have brought homologous recombinationand site—directed integration up to and beyond this level of efficiency．For all transgenesis techniquesin Drosophila melanogaster， microinjection of embryos is the central procedure．This chapter gives adetailed protocol for microinjection，and aims to enable the reader to use it for both site—directed inte—gration and for P—element transgenesis．　　Key words：Drosophila melanogaster，Embryo，Microinjection，Transgenic，Recombination，Inte—gration，Homologous recombination，phiC31／integrase，Site—directed integration，p—element　　1．1ntroduction　　Transgenesis in Drosophila melanogaster has undergone somethingof a revolution in the last few years．The classical technique ofrandom P—element—mediated transgenesis has recently been sup—plemented by two novel technologiCS：homologous recombi—nation and ΦC31 integration（for reviews，see（1）and（2）．InP—element transgenesis（3），a modified transposon vector is usedin combination witll transient expression of the P transposaseenzyme to generate several fly lines、with different insertion sitesin the genome．These insertions are subsequently mapped andcharacterised．P—element insertions have been invaluable formutagenesis screens，but until recently，this was also the only metllod available for introducing a transgene of choice into theDrosophila genome．The random nature of P—element insertaonshas sevcral drawbacks for transgene analysis．Mapping of inser—tion sites iS time consuming、and transgene expression levels aresubject to genomic position effects，making it difficult to drawcomparisons between different constructs．　　A recently developed alternative to random insertion ishomologous recombination（4，5）．This involves inserting adonor construct at random into the genome by P—element trans—genesis，and in subsequent generanons，mobilising the donorconstruct to the correct locus by homologous recombination．This technique had long been lacking to Drosophilists，but hasnot replaced P—element transgenesxs as the method of choice torroutine transgene analysis．because both the cloning of donorconstructs and the generation of homologous recombinants aremore time consuming than for P—element transgenesis．　　Reccently，ΦC31 integration has been developed（6）． Thistechnique allows rapid and efficient generation of site—specificintegrants，and relies upon‘docking site’fly lines，which carrya single recognition site（attP）for the phage ΦC31 integraseenzyme．previously introduced into the genome by P—elementtransgenesis．A donor plasmid carrying a second recognition site（attB）and a source of integrase enzyme is used to generate thcs inwhich the donor plasmid docks to the genomlc site．Integrationevents are highly specific，as the attP site is 39 bp long and doesnot occur at random in the Drosophila genome．Many mappedand characterised docking site lines arc now available（see Note 1），and ΦC31 integration is rapidly becoming widely used for manytransgcnic atmlications．　　……

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　　《轉(zhuǎn)基因技術：原理與實驗方案（實驗室解決方案）（原著第3版）》特點：　　本書為Springer經(jīng)典實驗室指南系列《分子生物學方法》（Methodsin Molecular Biology）的分卷。　　第3版對制備轉(zhuǎn)基因動物的實驗室可提供全面指導，對進行與轉(zhuǎn)基因相關科學研究的實驗室亦可提供豐富、寶貴的知識和信息。

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