非洲爪蟾

Experimental methods


• If the effect of, say, the VP16 fusion is similar to the normal factor this shows that the normal factor must be an activator. • If the EnR version gives a normal phenotype then the transcription factor is likely to be a repressor.
Experimental methods

It is also possible to introduce genes by transgenesis. To make transgenic embryos the transgene DNA is added to sperm heads that have been decondensed in egg extract to make their own DNA accessible, and these are then injected into unfertilized eggs.
Experimental methods
As many developmentally active molecules have different effects at different times, it may be desirable to inhibit activity of the introduced gene product until a desired stage. A useful method for doing this for transcription factors involves adding the hormone-binding domain from the glucocorticoid receptor to the protein of interest. This then causes it to be sequestered in the cytoplasm by binding to the cytoplasmic protein hsp90.



重要的模式生物

严重缺陷：
•非洲爪蟾生命周期过长，从受精发育至成熟具生殖能力的 成蛙需时一年； •非洲爪蟾是异源四倍体生物，多数基因都存在4个拷贝， 很难进行遗传突变试验。
胚胎发育
Cleavage Blastula Gastrulation
Neurulation
Tailbud stage
Prelarval shape

Experimental methods
Until such time as a glucocorticoid, ususally dexamethasone, is added. As a lipid-soluble substance, dexamethasone can penetrate into the embryo and will bind to the receptor, liberating it from the hsp90, and allowing it to move to the nucleus where the transcription factor part of the molecule can exert its biological activity.
Experimental methods
• If it is desired to inhibit a maternally acting gene, then antisense deoxy-oligonucleotides are also effective. • It is often very important to be able to deplete the oocyte of a specific mRNA since it is during these stages that various maternal components are laid down that are essential to later, zygotic, development. • The antisense oligo is injected into the oocyte and, like the morpholinos, hybridizes to the target messages. But in this case the resulting RNA–DNA hybrid is a target for the nuclease RNAseH which destroys the message.

Experimental methods

In order to establish later developmental effects of these experiments it is then necessary to make the treated oocytes into embryos.
标志性研究

戈登先用紫外线照射爪蟾卵细胞，破坏其细胞核，然后取 爪蟾蝌蚪的肠上皮细胞、肝细胞、肾细胞等的细胞核，植 入上述处理过的卵细胞内，其中一少部分卵会开始分裂并 可发育至一定时期。利用蝌蚪小肠上皮细胞作为核供体， 通过连续核移植的办法，戈登成功获得了少量蝌蚪，其中 有几只成功发育成为成体爪蟾完成了世界上最早的克隆动 物的实验。这一结果轰动了科学界，充分证明了细胞核的 全能性，也开创了动物克隆的时代。
重要的模式生物


优点：
取卵方便：通过注射激素，可以随时诱导非洲爪蟾排卵， 不受季节限制，更可通过人工授精获得受精卵； 非洲爪蟾卵子和胚胎个体较大，直径可达1.4毫米，很方 便进行实验胚胎学研究，如显微注射、胚胎切割和移植 等； 早期胚胎发育很快，在24℃下2天左右就可以孵化成可以 自由游动的幼虫，便于进行研究； 非洲爪蟾较为活泼，易于饲养。
标志性研究

•直到20世纪90年代，人们以非洲爪蟾为模型进行实验， 找到了组织者中真正的作用分子：BMP信号抑制因子和 Wnt信号抑制因子

• 1962年，英国牛津大学的生物学家戈登利用非洲爪
蟾进行了一系列的核移植试验 ，以研究不同发育时期 胚胎细胞核的发育能力。此实验证明了细胞核的全能性， 完成了世界上最早的克隆动物的实验，也开创了动物克隆 的时代。
Model Organism
Xenopus Laevis
Outline



1、非洲爪蟾简介； 2、重要的模式生物; 3、胚胎发育-重点讲原肠胚； 4、Experimental Methods； 5、标志性研究-重点戈登核移植； 6、标志性研究； 7、提问环节
非洲爪蟾
学名：Xenopus laevis 属名：Xenopus 门：脊索动物门 (Phylum Chordata）
原肠胚





非洲爪蟾预定内胚层细胞内陷，形成 狭缝状胚孔内陷引发原肠形成。 原肠作用的下一个时期包括边缘区细 胞的内卷以及动物半球细胞的外包和 向胚孔处集中. 随着瓶状细胞进入胚胎内部，背唇被 后来内卷进入胚胎发育为头部中胚层 前体的细胞代替。 随着新细胞进入胚胎内部，囊胚腔被 挤压到与背唇相对的一侧。 最终，卵黄栓也被包入内部。
Experimental methods


When attempting to establish the function of any gene product in development it is necessary to know at least the following: • 1、the expression pattern; • 2、the biological activity; • 3、the effect of specific inhibition in vivo.
Biblioteka Baidu
亚门：脊椎动物亚门(Vertebrata)
纲：两栖纲（Amphibia）
目：无尾目 (Salientia)
科：负子蟾科(Pipidae)
1802年，法国博物学家杜丁依据巴黎自然历史博物馆保存 的一件标本第一次描述了非洲爪蟾这一物种



•为完全水生种 •广栖于淡水水域中，尤其喜好静止水域的环境 •初春至晚夏间为繁殖期，雌蛙每胎可产下2000颗以上的 卵 •由于没有舌头只能利用其前肢搅食水中的无脊椎动物， 如果食物过大，它就用后肢上的爪将其撕碎
标志性研究



• 1927—1930年，英国生物学家霍格本教授利用爪蟾研究 出进行早期妊娠检测的方法，即注射孕妇尿（其有效成分 为绒毛膜促性腺激素）； •从20世纪50年代开始，非洲爪蟾逐渐成为发育生物学研 究的模式动物。荷兰发育生物学家纽库普与法伯合作在 1956年发表的非洲爪蟾发育图谱与分期标准为推广这一模 型做出了重要贡献，至今仍被广泛使用； • 20世纪80年代中期，英国的施莱克和史密斯利用作为 胚胎材料发现了成纤维细胞生长因子和活性素具有诱导能 力。
有关研究
Experimental methods


•In Xenopus ，it is not practical to study the effects of mutating genes to inactivity, but for inhibition experiments there are three standard protocols which are very effective. • The most commonly used is the injection of antisense morpholino oligos into fertilized eggs.
Progesterone→coloring them with a vital dye→reimplanting→embryo
Experimental methods
• The third protocol involves the design of dominant negative versions of gene products which can specifically inhibit the normal protein on overexpression. • In Xenopus, particularly extensive use has been made of domain swapped versions of transcription factors. • The effector domain of the factor is replaced by a strong activator (e.g. VP16) or a strong repressor (e.g. engrailed repressor).
Experimental methods
• For biological activity measurement the material to be injected will usually be a mRNA made in vitro. • The RNA synthesis is performed using plasmids carrying promoters for RNA polymerases of bacteriophage such as Sp6, T3, or T7,and a poly(A) addition site to ensure in vivo addition of poly(A) to stabilize the message.