by-2细胞的培养与转化

BY-2 Cells: Culture and Transformation for Live Cell Imaging BASIC PROTOCOL 1 ESTABLISHING A BY-2 SUSPENSION CULTURE BY-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 (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。 Materials Calli of wild-type and/or transformed BY-2 cells (available from various laboratories) 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 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 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 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 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. Avoid passing anything (e.g., hands or sleeves) over the open flasks. 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 (usually 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. 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 to 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 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. 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 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 2 ROUTINE CULTURE OF BY-2 CELLS 常规的BY-2细胞培养 This protocol describes 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。 Materials 50-ml conical flasks containing 20 ml liquid BY-2 medium (see 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, sterileTrimmed 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 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 readily 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 (usually 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。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 be used to cut the tips, and cut tips are autoclaved as normal. For larger volumes of cells, use 100 ml BY-2 medium in a 250- ml flask. For 100-ml suspension 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 fresh medium. 4. Cover the new flask with a new square of aluminum foil and seal firmly. 5. Place cells in a shaking incubator set at 130 rpm and 25C with illumination of choice for 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 8 hr dark can also be used. After 7 days, cells should be passaged again. 2、用 BASIC PROTOCOL 4 基本操作4 STABLE 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 targeted 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 resistance). 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 manufacturers 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。 Materials YEB medium (see recipe) containing appropriate filter-sterilized bacterial selection antibiotic Agrobacterium tumefaciens transformed with vector containing appropriate GFP construct (e.g., strain GV3101:pMP90; Konez and Schell, 1986) transformed with another plasmid (e.g., pVKHI8En6, pBII21) which contains GFP and the insert of interest. 3-day-old wild-type BY-2 suspension culture (see Basic Protocol 1) Liquid BY-2 medium (see recipe), sterile Solid BY-2 medium (see recipe) plates with plant selectable antibiotic, 100 g/ml carbenicillin (see recipe), and 20 g/ml timentin (see recipe) Shaking incubator, 25C Trimmed 1-ml pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterile 5- and 10-cm petri dishes, sterile 1.5-ml microcentrifuge tubes, sterile Forceps, sterile 材料 YEB培养基（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 agrobacteria 1. Inoculate 5 ml YEB medium containing appropriate filter-sterilized bacterial selection antibiotic with a single colony of Agrobacterium tumefaciens transformed with a binary vector containing the appropriate GFP construct. Incubate 18 to 20 hr at 130 rpm and 25C. 转化的农杆菌的生长 1、接种农杆菌单克隆于5mlYEB培养基，培养基中含有合适的过滤灭菌的筛选抗生素， 单克隆的农杆菌中转化有，有合适GFP结构的双元载体，培养 18 to 20 hr 在 130 rpm，25 C. Transform BY-2 cells 2. Using a trimmed 1-ml pipet tip, transfer 1 ml of a 3-day-old wild-type BY-2 suspension culture to a sterile 5-cm petri dish. 3. Add 50 l overnight Agrobacterium culture (step 1) and mix very gently with pipet tip. 4. Seal plate with Parafilm and incubate 2 days at 25C in the dark without shaking. Alternatively, the plates may be wrapped in aluminium foil or placed in sealed boxes. 5. Add 1 ml fresh liquid BY-2 medium and collect the cells with a trimmed 1-ml pipet tip. Transfer cells to a sterile 1.5-ml microcentrifuge tube. Because the cell medium may have dried out during the 2-day incubation, 1 ml medium is added to facilitate collection of the cells. It may be necessary to divide cells between two tubes at this stage. 6. Allow BY-2 cells to settle to bottom of tube by gravity (2 min). 7. Remove excess medium and add 1 ml fresh liquid BY-2 medium. Resuspend cells by gently flicking the tube. 8. Repeat steps 6 and 7 two more times for a total of three washes. 转化BY-2细胞 2、用斜剪的1ml枪头吸取1ml3天的野生型BY-2悬浮培养物至灭菌了的5cm的培养皿上 3、加50ul隔夜培养的农杆菌培养物（步骤1）用枪头轻轻混匀。 4、用Parafilm封口膜封口，在25度的暗环境中培养两天，不要摇动。 可选的：用铝膜纸封口，或放入密封箱中 5、 加入1ml新鲜的BY-2液体培养基，并用斜剪的1ml枪头收集细胞。将细胞转移至灭菌 的1.5-ml微量离心管 因为细胞的培养基在两天的培养过程中可能变干了，加入1ml培养基是为了促进收 集细胞的。在这一步中可能会需要将细胞分入两个离心管中。 6、 让BY-2细胞在1.5-ml微量离心管中静置2min 7、 吸取多余的上部的培养基，并加入1ml新鲜BY-2液体培养基。轻轻弹离心管重悬浮 细胞。 8、 重复步骤6和7两次，总共要洗三次。 Plate BY-2 cells 9. Resuspend cells with 1 ml fresh liquid BY-2 medium and use trimmed 1-ml pipet tip to transfer cells to solid BY-2 medium plates with plant selectable antibiotic, 100 g/ml carbenicillin and 20 g/ml Timentin. 10. Gently rotate plate to spread cells over the surface of the solid medium. 11. Seal plate with Parafilm and incubate in the dark at 25C without shaking until micro calli appear (1 month). 12. Use sterile forceps to excise individual microcalli and plate them onto fresh solid BY-2 medium plates with plant antibiotic. Place a maximum of nine calli per 10-cm plate. Calli can be screened, after excision and before passaging, for fluorescence using an UV lamp for constructs based on GFP5 (Haseloff et al., 1997); calli that have been successfully transformed will fluoresce. 13. Passage calli three times, allowing a 1-month interval between each passage, before establishing suspension cultures. BY-2细胞铺板 9、用1ml新鲜的液体BY-2培养基重新悬浮细胞，并用斜剪的枪头将细胞转移至固体的 BY-2培养基上，培养基上含有植株筛选抗生素100 g/ml羧苄青霉素（见配方）和20 g/ml特美汀（见配方） 10、轻轻转动平板，让细胞在固体培养基的表面平铺。 11、用Parafilm封口膜封口，在暗环境25度中培养，不要摇动，直到小的愈伤组织出 现（1个月） 12、用灭菌锅的镊子切除单独的小的愈伤组织并放入新鲜的固体BY-2培养基中，培养 基中含有植株抗生素。最多放入九个愈伤组织在10cm的平皿中。 愈伤组织是可以被鉴别的，在切除下来后，传代培养之前，荧光可以用UV灯，成功转 化了的愈伤组织会发出萤光。 13、在建立悬浮培养前三次继代培养愈伤组织，每次继代培养中间隔一个月。 Each passaging step except the first (when the microcalli are too small to divide) should be carried out in duplicate to ensure that, should one plate become contaminated, there is still another stock plate available. These passages are carried out to ensure that the cells are stably transformed. 每一个传代的步骤除了第一步（愈伤组织太小了而不能分开），都应该按一式两份来操作，确 保一旦将一个平板被污染了，还有另外一个储存的平板可以提供。这样就能使细胞稳定的被转 化。 Figure 1.7.4 (A) DIC image of BY-2 suspension culture. Membranous structures, nuclei, and vacuole are clearly visible. (B) Cortical endoplasmic reticulum (ER) visualized with yellow fluorescent protein (YFP) targeted and retained in a BY-2 cell stable transformant. This construct, SpYFP-HDEL, is composed of a sporamin signal peptide (to target the protein to the ER lumen), YFP, and the tetrapeptide HDEL to retain the protein in the lumen of the organelle (Irons et al., 2003). (C) To v