BY2细胞的培养与转化.docx

1、BY2细胞的培养与转化 BY-2 Cells: Culture andTransformation for Live Cell ImagingBASIC PROTOCOL 1ESTABLISHING A BY-2 SUSPENSION CULTUREBY-2悬浮培养的建立This protocol describes the method to establish a BY-2 cell suspension from a BY-2 or another tobacco callus. Subculturing the cells is technically termed passaging

2、 (also see Basic Protocol 2). It is advisable to work in duplicate or even triplicate when establishing a suspension culture. This allows initiation of fresh suspensions of the same transformed cell line should any contamination occur.这个实验步骤描述了通过BY-2或者其他的烟草愈伤组织建立BY-2细胞悬液的方法。传代培养细胞是一个传代的专有名词。当建立悬浮体系的

3、时候最好是一式两份或一式三份的进行。这样就使得一旦一个转化的细胞株的污染发生的时候，又可以开始新的细胞悬浮。MaterialsCalli of wild-type and/or transformed BY-2 cells (available from variouslaboratories) grown on solid BY-2 medium (see recipe) in petri dish 50-ml conical flasks containing 20 ml liquid BY-2 medium (see recipe), covered with aluminum foil

4、 and autoclaved Suitable filter-sterilized antibiotics, for culturing transformed BY-2 cells only Razor blades, sterile Packet of aluminum foil squares for covering flasks, sterile Shaking incubator, 25C 1. Cut a 2-cm2 piece of wild-type or transformed BY-2 callus with a sterile razor blade directly

5、 on the petri dish on which the calli are growing.2. Loosen aluminium foil from top of a sterile 50-ml conical flask containing 20 ml liquid BY-2 medium. Lightly flame neck of flask. Add suitable filter-sterilized antibiotics if needed. Open a sterile packet of aluminum foil squares so that they are

6、 readily accessible.材料转化过的或野生型的BY-2细胞的愈伤组织（不同的实验室都有）生长于培养皿的BY-2固体培养基上（见配方）装有20ml液体BY-2培养基（见配方）的三角瓶，用铝膜纸封口高温灭菌。过滤灭菌-转化BY-2细胞相关的抗生素无菌的保险刀片一捆方形的铝膜纸用来包烧瓶的，灭菌25度摇床1、 直接在培养基上用灭菌的刀片切2平方厘米的野生型或者是转化的BY-2愈伤组织，并让其在培养基上生长。2、 松开灭菌的装有液体培养基的三角瓶的铝膜封口，用火烧瓶口，加入合适的抗生素。打开一个正方形的铝膜纸，先做好准备。This will aid the speed at which

7、 the items in the hood can be handled and hence will reduce the possibility of contamination.The flasks should be open for as short a time as possible to avoid contamination. Avoidpassing anything (e.g., hands or sleeves) over the open flasks.The preparation and storage of antibiotics should be acco

8、rding to manufacturers instructions. Antibiotics should be filter sterilized and handled as sterile material. To filter sterilize the antibiotic solution, a stock solution of the required concentration (usually 100) is prepared. The antibiotic solution is aspirated into a sterile syringe without a n

9、eedle. A 0.2-_m syringe filter is attached to the syringe, and the solution is pressed through the filter into a sterile tube.这样可以加快在工作台上的实验速度继而降低污染的可能性。烧瓶的打开时间应该尽可能的的短来避免污染。避免任何东西经过打开的烧瓶（如，手或袖子）抗生素的准备和存放应该遵守抗生素制造商的说明书。抗生素应该用过滤灭菌，而且用灭菌过的材料来存放。准备的母液浓度一般是100X。抗生素溶液用灭菌了的注射器吸上来。0.2-_m的注射器式灭菌器装在注射器上以后，推注

10、射器，将经过过滤器的溶液装入灭菌了的管中。3. Transfer the cut callus to the 50-ml flask with medium.将切下来的愈伤组织转移至装有50ml培养基的烧瓶中。 When establishing suspension cultures it is best to begin in a small volume as the cells grow better。T encourage growth the authors have found that subculturing newly established suspensions 1 t

11、o 2 weeks after placing calli in liquid medium, the culture should be quite thicklike runny tomato ketchup. Passaging cultures at lower dilution ratio (1:10 instead of 1:20) can also encourage good suspension culture growth. Once the cells are growing well in the small volume they can be subcultured

12、 into larger volumes of medium. 当建立了悬浮培养时，最好是先从小的容积开始，这样细胞会长得好一些。为了使细胞生长得更好，作者发现新的继代悬浮培养是在将愈伤组织放入液体培养基后的1-2周。培养物应该是非常浓稠的，像半液体状的番茄酱。继代培养在一个低一点的稀释比（1:10而不是1:20）可以使悬浮培养生长得更好。一旦细胞在小的容器中长得很好时，就可以将其继代培养转入大一些的装有培养基的器中了。4. Gently pipet the culturing medium up and down to break up callus. 轻轻的上下吸打培养基，打散愈伤组织。5

13、. Reseal the flask with a new square of aluminium foil. 用新的方形的铝膜纸重新密封烧瓶。6. Place cells in a shaking incubator set at 130 rpm and 25C with illumination of choice.将细胞放在摇床中，设置130rpm，25度，光照设置是可选择的A flat-bed orbital platform in a 25C culture room may also be used.BY-2 cell suspensions may be kept in the

14、dark. An illumination regime of 16 hr light and 8 r dark can also be used.Cells should be subcultured after 7 days (see Basic Protocol 2).一个平的板在25度的培养空间是需要的，BY-2细胞的悬浮培养需要在黑暗的环境中，或者是16小时的光照，8小时的黑暗也是可以的。细胞的继代培养要在7天以后（见基本操作2）BASIC PROTOCOL 2ROUTINE CULTURE OF BY-2 CELLS常规的BY-2细胞培养This protocol describe

15、s the routine method to culture BY-2 cells and to maintainsuspension and callus cultures. As described in Figure 1.7.1, the protocol requires liquid andlid media 这个操作方法描述了常规的培养BY-2的方法和保持继代和愈伤组织的培养。像如1.7.1描述的一样，这个操作守则需要液体的还有固体的培养基。Materials50-ml conical flasks containing 20 ml liquid BY-2 medium (see

16、 recipe), coveredwith aluminum foil and autoclavedSuitable filter-sterilized antibiotics, for transformed BY-2 cells onlyWild-type or transformed stationary-phase BY-2 cells (i.e., 7-day-old cultures)grown in suspension (see Basic Protocol 1)Packet of aluminum foil squares for covering flasks, steri

17、leTrimmed 1-ml pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterileShaking incubator, 25C材料50ml的三角瓶装有20ml的液体的BY-2液体培养基，用铝膜纸封住，高温灭菌。抗体，专门用来转化BY-2细胞的，过滤灭菌。野生的或转化的，生长于悬浮液的稳定的BY-2细胞，（例如。7天的培养物）（见基本操作1）一捆方形的铝膜纸用来包烧瓶的，灭菌斜剪好的枪头灭菌，从枪头的前端4-5毫米处斜剪，25度的摇床。1. Loosen aluminium foil from top of a sterile

18、 50-ml conical flask containing 20 mlliquid BY-2 medium. Lightly flame neck of flask. Add suitable filter-sterilizedantibiotics if needed. Open a sterile packet of aluminum foil squares so that they arereadily accessible.3、 1、松开灭菌的装有20mlBY-2液体培养基的50ml三角瓶的铝膜封口，用火烧瓶口，加入合适的抗生素。打开一个正方形的铝膜纸，先做好准备。图A为常规培养

19、BY-2细胞的方法（参见操作方法2）图B稳定转化子的产生（参见操作方法4）The preparation and storage of antibiotics should be according to manufacturers instructions. Antibiotics should be filter sterilized and handled as sterile material. To filter sterilize the antibiotic solution, a stock solution of the required concentration (usu

20、ally 100) is prepared. The antibiotic solution is aspirated into a sterile syringe without a needle. A 0.2-_m syringe filter is attached to the syringe, and the solution is pressed through the filter into a sterile tube.抗生素的准备和保存应该遵循生产商的说明。抗生素需要被过滤灭菌并被保存在灭菌的容器中。用来过滤灭菌的溶液，通常的储存浓度是100。抗生素溶液用灭菌了的注射器吸上来

21、。0.2-_m的注射器式灭菌器装在注射器上以后，推注射器，将经过过滤器的溶液装入灭菌了的管中。2. Use a trimmed 1-ml pipet tip to take a 1-ml aliquot of wild-type or transformed stationary-phase BY-2 cells from their flask.The ends of uncut tips will be too narrow to allow easy entry of cells. A razor blade can beused to cut the tips, and cut tip

22、s are autoclaved as normal.For larger volumes of cells, use 100 ml BY-2 medium in a 250-ml flask. For 100-mlsuspension cultures, passage 10 ml cells into 100 ml fresh medium.3. Remove foil from the new flask and pipet the cell aliquot directly into the freshmedium.4. Cover the new flask with a new s

23、quare of aluminum foil and seal firmly.5. Place cells in a shaking incubator set at 130 rpm and 25C with illumination of choicefor 7 days.A flat-bed orbital platform in a 25C culture room may also be used.BY-2 cell suspensions may be kept in the dark. An illumination regime of 16 hr light and 8hr da

24、rk can also be used.After 7 days, cells should be passaged again.2、用BASIC PROTOCOL 4基本操作4STABLE TRANSFORMATION OF BY-2 CELLS MEDIATED BY AGROBACTERIUM FOR VISUALIZATION OF SUBCELLULAR ORGANELLES由农杆菌介导的亚细胞器自显的BY-2细胞的稳定转化This protocol involves the production of BY-2 lines that stably express GFP targe

25、ted to subcellular structures for subsequent live cell imaging (see Fig. 1.7.3). The procedures are suitable for other tobacco cell lines and may be modified for Arabidopsis thaliana suspension cells as well这个操作为构建可以在BY-2细胞中目标亚细胞结构中稳定表达的GFP，并在实时亚细胞图片观察中能看到的。（见图1.7.3）这个操作也适合其他的烟草细胞系，在改良后也可以适用于拟南芥的悬浮细

26、胞。NOTE: To transform BY-2 cells, a binary vector that can be transformed into agrobacteria is needed and can be the same as is used to transform tobacco and Arabidopsis plants. The binary vector, in which the fluorescent protein marker is subcloned, must carry two resistance markers (antibiotic resi

27、stance). One is a selectable marker for bacteriato select positive transformants after bacterial transformationand the other is a selectable marker for plants, which will allow the growth only of transformed plant cells. When working with these selection markers, it is very important to follow the m

28、anufacturers instructions for the specific antibiotic in use. Factors such as light and pH, for example, may alter the properties of the antibiotics and thus affect the yield of stable transformants.注意：为了转化BY-2细胞，需要一个二元的能转入农杆菌的载体，同时也能一样转入烟草和拟南芥的植株中。这个二元载体，必须有荧光蛋白标记的亚克隆，同时带有两个抗体标记。一个是细菌筛选标记-在细菌转化后用来筛

29、选阳性的转化细胞；另一个是植株的筛选标记，使得只有在转化了的植株细胞才能生长。当使用这些筛选标记时，根据制造商的说明来使用特定的抗生素是很重要的。一些影响因子，例如光照和PH值，可能会改变抗生素的性能，从而影响到转化细胞产量的稳定性。MaterialsYEB medium (see recipe) containing appropriate filter-sterilized bacterialselection antibioticAgrobacterium tumefaciens transformed with vector containing appropriate GFPcons

30、truct (e.g., strain GV3101:pMP90; Konez and Schell, 1986) transformedwith another plasmid (e.g., pVKHI8En6, pBII21) which contains GFP and theinsert of interest.3-day-old wild-type BY-2 suspension culture (see Basic Protocol 1)Liquid BY-2 medium (see recipe), sterileSolid BY-2 medium (see recipe) pl

31、ates with plant selectable antibiotic, 100 g/mlcarbenicillin (see recipe), and 20 g/ml timentin (see recipe)Shaking incubator, 25CTrimmed 1-ml pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterile5- and 10-cm petri dishes, sterile1.5-ml microcentrifuge tubes, sterileForceps, sterile材料YEB培养基（

32、见配方）含有过滤灭菌后的细菌筛选抗生素转化过的农杆菌，转化的载体中含有合适的GFP结构（例如：菌株 GV3101:pMP90; Konez and Schell, 1986）转化的其他质粒（例如：pVKHI8En6, pBII21），其中也包含GFP和插入片段。3天的野生BY-2悬浮培养物（见基本操作1）液体BY-2培养基（见配方），灭菌固体BY-2培养基（见配方），加入植株筛选抗生素100 g/ml羧苄青霉素（见配方）和20 g/ml特美汀（见配方）25度摇床斜剪的1-ml枪头（从枪头的前端4-5毫米处斜剪）灭菌5- 和 10-cm的培养皿，灭菌1.5-ml微量离心管，灭菌镊子，灭菌Grow agrobacteria1. Inoculate 5 ml YEB medium containing appropriate filter-sterilized bacterial selection antibiotic with a sin