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在后基因组时代，学界所面临的主要挑战之一是如何破解大量的编码蛋白质的基因功能。在《转基因技术：原理与实验方案》第3版中，该领域的专家在第2版的基础上进行了更新和补充，以求能够详尽地反映当前基因修饰技术的*进展。本书不仅包括基因修饰小鼠制作过程，同时也介绍了其他模式生物的转基因技术，以及在显微注射、位点特异性重组系统、冷冻保存等方面的探索和尝试。本书秉承Springer《分子生物学方法》系列丛书的一贯风格，阐述明晰、便于使用，每章包括对相关问题的介绍，所需材料和试剂的清单，实验操作的具体步骤，以及常见问题的解决方法和缺陷规避。

前言

撰稿人

第一部分 多种模式动物的转基因技术

1 转基因果蝇

2 转基因线虫

3 利用Tol2转座子系统制作转基因斑马鱼

4 转基因爪蟾

5 利用DNA前核注射制作转基因大鼠

第二部分 小鼠的转基因技术

6 细胞类型特异的转基因小鼠

7 设计转基因载体和DNA转入基因组-成功的关键

8 过表达转基因-前核显微注射

9 基因打靶载体

10 基因捕获:基因敲除的快速通道

11 小鼠胚胎干细胞的培养

12 打靶胚胎干细胞

13 通过显微注射获得嵌合体小鼠

14 通过桑葚胚细胞聚集获得嵌合体小鼠

15 获得突变体小鼠相关的手术操作

16 用于小鼠基因组修饰相关的位点特异性重组酶

17 Cre转基因小鼠

18 大规模小鼠基因突变

19 小鼠的繁殖和饲养

20 维持和保存实验室变异小鼠的生物学方法Ⅰ:保存突变小鼠株

21 维持和保存实验室变异小鼠的生物学方法Ⅱ:复苏突变小鼠株

索引

Cardiov ascular Medicine，University of Manchester，Manchester，UK

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Chapter 1

Transgenesis in Drosophila melanogaster

Leonie Ringrose

Summary

Transgenesis in Drosophila melanogaster relies upon direct microinjection of embryos and subsequent crossing of surviving adults. The necessity of crossing single flies to screen for transgenic events limits the range of useful transgenesis techniques to those that have a very high frequency of integration, so that about 1 in 10 to 1 in 100 surviving adult flies carry a transgene. Until recently, only random P-element transgenesis fulfilled these criteria. However, recent advances have brought homologous recombination and site-directed integration up to and beyond this level of efficiency. For all transgenesis techniques in Drosophila melanogaster, microinjection of embryos is the central procedure. This chapter gives a detailed 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. Introduction

Transgenesis in Drosophila melanogaster has undergone something of a revolution in the last few years. The classical technique of random P-element-mediated transgenesis has recently been sup-plemented by two novel technologies: homologous recombi-nation and ΦC31 integration (for reviews, see (1) and (2)). In P-element transgenesis (3), a modified transposon vector is used in combination with transient expression of the P transposase enzyme to generate several fly lines with different insertion sites in the genome. These insertions are subsequently mapped and characterised. P-element insertions have been invaluable for mutagenesis screens, but until recently, this was also the only

Elizabeth J. Cartwright (ed.), Transgenesis Techniques, Methods in Molecular Biology, vol. 561 DOI 10.1007/978-1-60327-019-9_1, . Humana Press, a part of Springer Science + Business Media, LLC 2009

3

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method available for introducing a transgene of choice into the Drosophila genome. The random nature of P-element insertions has several drawbacks for transgene analysis. Mapping of inser-tion sites is time consuming, and transgene expression levels are subject to genomic position effects, making it difficult to draw comparisons between different constructs.

A recently developed alternative to random insertion is homologous recombination (4, 5). This involves inserting a donor construct at random into the genome by P-element trans-genesis, and in subsequent generations, mobilising the donor construct to the correct locus by homologous recombination. This technique had long been lacking to Drosophilists, but has not replaced P-element transgenesis as the method of choice for routine transgene analysis, because both the cloning of donor constructs and the generation of homologous recombinants are more time consuming than for P-element transgenesis.

Recently, ΦC31 integration has been developed (6). This technique allows rapid and efficient generation of site-specific integrants, and relies upon ‘docking site’ fly lines, which carry a single recognition site (attP) for the phage ΦC31 integrase enzyme, previously introduced into the genome by P-element transgenesis. A donor plasmid carrying a second recognition site (attB) and a source of integrase enzyme is used to generate flies in which the donor plasmid docks to the genomic site. Integration events are highly specific, as the attP site is 39 bp long and does not occur at random in the Drosophila genome. Many mapped and characterised docking site lines are now available ( see Note 1), and ΦC31 integration is rapidly becoming widely used for many transgenic applications.

All these transgenic techniques rely upon microinjection of embryos as a first step. In early Drosophila embryogenesis, the nuclei share a common cytoplasm for the first nine divisions. Directly after the tenth division, the first cells to become sep-arated are the pole cells, which will later form the adult germ line. Transgenic animals are made by microinjecting DNA and a source of enzyme (P-transposase or ΦC31 integrase, see Note 2) into the posterior of the embryo where the pole cells will form, at an early stage before they have become separated from the common cytoplasm. DNA can enter the nuclei and is integrated into the genome of some cells. Embryos are allowed to mature and the adults are outcrossed to screen for transgenic flies in the next generation.

This chapter gives a detailed description of microinjection, from preparing DNA to screening for transformants. The main protocol deals with ΦC31 integration as we perform it in our laboratory. Alternatives for both ΦC31 and P element transgen-esis are given in the notes.

Transgenesis in Drosophila melanogaster

2. Materials

2.1. Preparation of DNA

2.2. Preparation of Injection Needles (see Note 4 )

2.3. Preparation of Flies for Egg Laying

2.4. Dechorionation and Dessication of Embryos

1.

Donor plasmid containing attB site and transgene of interest (see Note 3).

2.

Helper plasmid expressing ΦC31 integrase (see Note 2). Midi- or miniprep kit for preparation of plasmid DNA (Qiagen).

3.

Absolute ethanol.

4.

3 M NaOAc, pH 5.2.

5.

Sterile distilled water.

1.

Capillaries: borosilicate glass capillaries, 1.2 mm × 0.94 mm

2.

Needle puller: P-97 micropipette puller (Sutter instruments).

3.

Needle grinder: Narishige microgrinder EG-400.

1.

Fly line containing genomic attP site (see Note 1).

2.

Fly bottles.

3.

Fresh yeast paste: cubes of fresh baker’s yeast cubes are obtain-able from large supermarkets. They can be frozen and stored at .20°C for several months. Thaw at room temperature and mix with a little water to give a thick paste.

4.

Dried yeast: Mix instant yeast granules with water to give a thick paste. Both fresh and dried yeast paste can be kept at 4°C for up to a week. Do not seal the container tightly, as the paste will expand.

5.

Fly cages: PVC plastic tubing of either 50 mm or 90 mm diameter is cut into 100?150 mm sections and sealed at one end with nylon or metal mesh. The other end fits onto to a 50-mm or 90-mm agar plate, which is taped in place for egg collection.

6.

Agar plates: Add 18 g agar to 600 mL tap water and bring to boiling point by microwaving. Dissolve 10 g sucrose in 300 mL tap water, heating a little if necessary. Add the sucrose solution to the agar, add 3.5 mL 100% acetic acid and mix well. Pour into petri dishes (90 mm or 50 mm) and allow to cool. Store for 1 day at room temperature to dry before using. Plates can be stored wrapped in plastic at 4°C for several weeks. About 16?20 plates per day of injection are required per cage (see Note 5).

1. Filtration apparatus consisting of glass funnel, filter support, stopper, sidearm flask, and clamp, suitable for 50-mm mem-brane filters. Attach the apparatus to water tap as shown in Fig. 1.

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Fig. 1. Filtration apparatus.

2.

Bleach solution: mix 50 mL household bleach (2.8% hypochlorite) with 50 mL sterile distilled water. Make fresh every day. Wear a lab coat and gloves when handling bleach, as it bleaches clothes upon contact and is harmful to skin.

3.

Membrane filters: mixed cellulose ester membrane filters, black with white grid marking. Circular, 50-mm diameter, 0.6- μm pore size (Schleicher and Schuell, type ME 26/31 ST).

4.

Binocular dissection microscope.

5.

Fine stiff paintbrush with nylon hairs: cut away hairs until only a few remain, for use in aligning embryos.

6.

Dissection needle.

7.

Forceps.

8.

Microscope slides: use slides with frosted part for labelling, such as Superfrost plus (Fisher).

9.

Coverslips: 24 × 24 mm.

10.

Embryo glue: Make three balls of 2.5-m Scotch tape Magic 810 (3 M). Add these to 30 mL heptane in a 50-mL falcon tube. Shake vigorously at 28°C for 24 h. Cut a hole in the bottom of the falcon tube and drain solution into a small glass bottle. This glue keeps for several months at room tem-perature ( see Note 6).

11.

Drying chamber: 150-mm petri dish containing orange self-indicating silica gel granules: check that the silica gel gran-ules are orange; if they are not then they are saturated and no longer effective for drying embryos. Change to fresh granules.

12.

Halocarbon oil: Voltalef 10S halocarbon oil, or halocarbon 700 oil (Sigma).

2.5. Microinjection of Embryos

2.6. Further Handling and Screening for Transgenics

Transgenesis in Drosophila melanogaster

1.

Microscope: Either a compound or inverted microscope is suitable for injection. We use a Zeiss Axiovert 200 inverted microscope with ×10 objective and ×10 oculars.

2.

Micromanipulator and needle holder (Narishige).

3.

Microinjection system: Femtojet 5247 programmable microin-jector with integrated pressure supply (Eppendorf) ( see Note 7).

4.

Microloader pipette tips (Eppendorf).

1.

Humid box: sealable plastic sandwich box containing damp paper towels.

2.

50-mm Petri dishes.

3.

18 mm × 18 mm cover slips.

4.

Flies for crossing to surviving adults: w- or appropriate bal-ancer lines.

5.

Fly vials.

3. Methods

3.1. Preparation of DNA

3.2. Preparation of Injection Needles

1.

Prepare donor and helper plasmids in advance. Use midi- or miniprep (Qiagen quality) DNA. Do not elute the DNA in the buffer provided, as it contains Tris buffer, which is harmful to embryos. Instead, elute in sterile distilled water ( see Note 8).

2.

Check the concentration of eluted DNA. If the concentration is sufficient, make an injection mix at 250 ng/ μL of donor vector plus 600 ng/μL of helper, in sterile distilled water (see Note 9).

3.

If the DNA concentration is too low, precipitate the DNA: Add

0.1 volume of 3 M NaOAc, pH 5.2, and two volumes of abso-lute ethanol. Incubate at ?20°C overnight. Centrifuge at 4°C for 10 min at 14,000 × g. Remove the supernatant, add 70% ethanol to the pellet, and centrifuge at 4°C for 5 min at 14,000 × g. Air dry the pellet and resuspend in sterile distilled water.

4.

Plasmids and injection mixes can be stored indefinitely at .20°C. For DNA stored in water, however, the absence of a buffering agent may lead to degradation upon repeated freez-ing and thawing (see Note 8).

1. Before beginning to inject, prepare a supply of needles. We use a needle puller (P-97, Sutter instruments) with the follow-ing settings: Heat = 595; Pull = 70; Vel = 80). Insert a glass capillary into the needle puller, close the lid, and press ‘pull’. This makes two needles from each capillary that are closed at the tapered end ( see Note 4).

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3.3. Preparation of Flies for Egg Laying

3.4. Dechorionation, Lining Up, and Dessi-cation of Embryos

2. Open the needles by grinding in the needle grinder. Insert the needle into the holder at an angle of 40° to the grind-stone. Keeping a constant flow of water over the grindstone, lower the needle onto the grindstone till the tip bends very slightly and water rises up into the needle. Immediately the water enters; stop moving the needle and allow to grind for 20 s (see Note 9).

1.

Expand the fly line that is to be injected to give six bottles. Flip all six each week if large-scale injections are planned. Use flies that are 1-week old and well fed for the best egg laying.

2.

One week before injection: flip adult flies every 2 days into bottles with fresh yeast paste. This feeds them optimally, so females lay a lot of eggs. Keep these bottles at 18°C.

3.

Two days before injection: transfer flies to cages (use 4?6 bot-tles per 90-mm-diameter cage). Add a little dried yeast paste on a small square of paper (this facilitates later removal) onto the plates and place the cages at 25°C. Change the plates every 24 h and discard them. This acclimatises flies to the cage envi-ronment.

4.

On the day of injection: Ensure that plates are at room tem-perature. Change the overnight plate, and wipe the inner rim of the cage to remove any first instar larvae. Add a very small spot of yeast paste on a square of paper to the centre of each new plate. Change the first plate after 1 h, and discard it. This is because females may keep fertilised eggs for some time before laying them. Use the subsequent plates for collections.

5.

Change the plates every 30 min to ensure that embryos can be collected, prepared, and injected before the germ cells form. For an optimal injection workflow, flies should be laying about 200 eggs every 30 min.

1.

Change the plate after 30 min laying, and remove the yeast and paper square. Set a timer for 2 min. Add bleach solution directly onto the plate and incubate for 2 min. Wear a lab coat to protect clothing from bleach.

2.

Assemble the filtration apparatus with a fresh membrane filter as shown in Fig. 1. Clamp the apparatus together. Add water and filter through to wet the membrane. Start the tap and open the screw on the sidearm flask. After exactly 2 min, tip the bleach from the plates onto the filter. Close the screw just until the liquid goes through, and then open it again, to avoid damaging embryos.

3.

Add water to the plates and filter in the same way. Add water to the filter and filter through. Always be aware that too much suction will damage embryos: open the screw on the sidearm flask as soon as the liquid goes through the membrane. Remove

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编辑推荐

　　卡特莱特编写的《转基因技术(原理与实验方案原著第3版导读版)(精)》用大部分的章节介绍了通过将外源DNA注射入受精卵的前核和基于在胚胎干细胞进行同源重组打靶技术的基因修饰小鼠的制作，该部分内容是专门为这本新版《转基因技术：原理与实验方案》编写的，涵盖了该领域内的**进展。主要介绍了构建转基因载体的**方案和策略、详尽的前核显微注射技术及相关的外科手术操作、维持胚胎干细胞多能性的**培养条件以及打靶技术等。第13—15章重点阐述了如何通过将打靶后的胚胎干细胞注射人囊胚或在桑葚胚阶段通过细胞聚集获得嵌合体小鼠的技术手段、将改造后的胚胎重新转入代孕母鼠体内的外科手术。多年来，Cre／loxP和flp／frt重组系统一直很受人们的欢迎，在16章对这两个系统及其在小鼠基因组修饰操作中的应用进行了介绍。17章重点介绍了Cre转基因小鼠的制作和应用。另有一章介绍了大规模的国际间合作以求在基因组中系统性敲除每个基因的工作。本书的其他章节介绍了成功增殖转基因小鼠所必备的饲养和繁殖技巧、冰冻保存以及从冰冻状态下复苏小鼠的技术。

书籍介绍

在后基因组时代，学界所面临的主要挑战之一是如何破解大量的编码蛋白质的基因功能。在《转基因技术:原理与实验方案(原著第3版)(导读版)》中，该领域的专家在第2版的基础上进行了更新和补充，以求能够详尽地反映当前基因修饰技术的最新进展。《转基因技术:原理与实验方案(原著第3版)(导读版)》不仅包括基因修饰小鼠制作过程，同时也介绍了其他模式生物的转基因技术，以及在显微注射、位点特异性重组系统、冷冻保存等方面的探索和尝试。