ES-cellen: möjligheter att manipulera det tidiga embryot

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1 Seminarium VBE ES-cellen: möjligheter att manipulera det tidiga embryot Seminariet syftar till att belysa de möjligheter som olika gentekniker ger för att manipulera embryonala stamceller men seminariet syftar också till att belysa och öva diskussion av etiska frågor som uppkommer till följd av genteknikerna. Som stöd bifogas sidhänvisningar, länkar till en film, respektive till CODEXhemsida, frågeställningar och förslag till diskussionspunkter. Det förutsätts att materialet är genomgånget och frågorna är bearbetade av studenterna innan seminariet. Studentresurser Lodish et al. (6:e upplagan): sid , och (7:e upplagan): sid och Larssen sid , 162 Alberts et al. (5:e upplagan): sid (bifogas) Film: namn:embryo lösenord:emb - Titta på de fem avsnitten! CODEX regler och riktlinjer för forskning:

2 1. Följande begrepp används i filmen IWF C 1665 Fortplantningens bioteknik: Mikromanipulation av äggceller och tidiga embryon (mus) : Gå igenom dessa i gruppen vid seminariets början! Ovulation Zona pellucida Ooplasma Hyaluronidas Pronuclei Polkropp Perivitellinutrymme Zygot Delning (klyvning) Blastomer, blastomerer Kompaktering EDTA Blastocyst Yttre och inre cellmassa Trofoblast Totipotens och pluripotens Kläckning Implantation 2. Vad är regulationsutveckling? (Jämför med mosaikutveckling) Hur kan man skapa aggregationschimärer eller erhålla flera individer ur ett enda tidigt embryo? 3. Diskutera i gruppen hur man kan skapa transgena djur med hjälp av pronukleär injektion. Hur kan man skapa transgena djur med hjälp av genetiskt manipulerade embryonala stamceller? Hur går man vidare med de chimärer som erhålls? 4. Vad är reproduktiv respektive terapeutisk kloning? Vad kan dessa tekniker användas till? 2

3 5. Diskutera hur dessa tekniker kan användas för människan (humanmedicinska applikationer)! I vilka fall anser ni det etiskt försvarbart att isolera celler från 4- till 8-cellsstadiet för preimplantorisk genetisk diagnostik? Kan möjligheterna att överföra gener till det tidiga embryot ge upphov till etiska problem? Finns det några etiska dilemman med att utvinna stamceller från navelsträngsblod/benmärg från t.ex. ett nyfött barn för att behandla ett sjukt syskon? Vad anser ni om marknadsförning av nedfrysning av stamceller från navelsträngsblod riktat mot föräldrar att använda om/när deras barn drabbas av sjukdom eller olycka (exempelvis danska företaget Stemcare )? Finns det någon moralisk skillnad i att utföra terapeutisk kloning med ES-celler från aborterat foster jämfört med ES-celler med ursprung hos endogen somatisk cell? Skulle det kunna vara etiskt försvarbart att utföra reproduktiv kloning? 3

4 Utdrag ur Alberts et al., 5:e upplagan (Molecular Biology of the Cell), sid : The Early Mammalian Embryo Is Highly Regulative Localized intracellular determinants play only a small part in early mammalian development, and the blastomeres produced by the first few cell divisions are remarkably adaptable. If the early embryo is split in two, a pair of identical twins can be produced two complete normal individuals from a single cell. Similarly, if one of the cells in a 2- cell mouse embryo is destroyed by pricking it with a needle and the resulting half- embryo is placed in the uterus of a foster mother to develop, in many cases a perfectly normal mouse will emerge.conversely, two 8- cell mouse embryos can be combined to form a single giant morula, which then develops into a mouse of normal size and structure (Figure 22 90). Such creatures, formed from aggregates of genetically different groups of cells, are called chimeras. Chimeras can also be made by injecting cells from an early embryo of one genotype into a blastocyst of another genotype. The injected cells become incorporated into the inner cell mass of the host blastocyst, and a chimeric animal develops. A single cell taken from an 8- cell embryo or from the inner cell mass of another early blastocyst can give rise in these ways to any combination of cell types in the chimera. Wherever the added cell may happen to find itself, it responds correctly to cues from its neighbors and follows the appropriate developmental pathway. These findings have two implications. First, during the early stages, the developmental system is self- adjusting, so that a normal structure emerges even if the starting conditions are perturbed. Embryos or parts of embryos that have this property are said to be regulative. Second, the individual cells of the inner cell mass are initially totipotent, or very nearly so: though they cannot form trophoblast, they can give rise to any part of the adult body, including germ cells. Figure A procedure for creating a chimeric mouse. Two morulae of different genotypes are combined. 4

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6 Totipotent Embryonic Stem Cells Can Be Obtained From a Mammalian Embryo If a normal early mouse embryo is grafted into the kidney or testis of an adult, its development is disturbed beyond any possibility of proper regulation, but not halted. The result is a bizarre tumorous growth known as a teratoma, consisting of a disorganized mass of cells containing many varieties of differentiated tissue skin, bone, glandular epithelium, and so on mixed with undifferentiated stem cells that continue to divide and generate yet more of these differentiated tissues. Investigation of the stem cells in teratomas and related types of tumors led to the discovery that their behavior reflects a remarkable property of the cells of the normal inner cell mass: given a suitable environment, they can be induced to proliferate indefinitely while retaining their totipotent character. Cultured cells with this property are called embryonic stem cells, or ES cells. They can be derived by placing a normal inner cell mass in culture and dispersing the cells as soon as they proliferate. Separating the cells from their normal neighbors and putting them in the appropriate culture medium evidently arrests the normal program of change of cell character with time and so enables the cells to carry on dividing indefinitely without differentiating. Many tissues of the adult body also contain stem cells that can divide indefinitely without terminally differentiating, as we shall see in the next chapter; but these adult stem cells, when allowed to differentiate, normally give rise only to a narrowly restricted range of differentiated cell types. The state in which the ES cells are arrested seems to be equivalent to that of normal inner- cell- mass cells. This can be shown by taking ES cells from the culture dish and injecting them into a normal blastocyst (Figure 22 91). The injected cells become incorporated in the inner cell mass of the blastocyst and can contribute to the formation of an apparently normal chimeric mouse. Descendants of the injected stem cells can be found in practically any of the tissues of this mouse, where they differentiate in a well- behaved manner appropriate to their location and can even form viable germ cells. The extraordinarily adaptable behavior of ES cells shows that cues from a cell s neighbors not only guide choices between different pathways of differentiation, 6

7 but can also stop or start the developmental clock the processes that drive a cell to progress from an embryonic to an adult state. Figure Making a chimeric mouse with ES cells. The cultured ES cells can combine with the cells of a normal blastocyst to form a healthy chimeric mouse, and can contribute to any of its tissues, including the germ line. Thus the ES cells are totipotent. 7

8 On a practical level, ES cells have a twofold importance. First, from a medical point of view, they offer the prospect of a versatile source of cells for repair of damaged and defective tissues in the adult body Second, ES cells make possible the most precisely controlled forms of genetic modification, allowing animals to be created with virtually any desired alteration introduced into their genome. The technique uses genetic recombination to substitute an artificially constructed DNA segment for the normal DNA sequence at a chosen site in the genome of an ES cell. Although only a rare cell incorporates the DNA construct correctly, selection procedures have been devised to find this cell among the thousands of cells into which the DNA construct has been transfected. Once selected, the genetically modified ES cells can be injected into a blastocyst to make a chimeric mouse. This mouse will, with luck, have some ES- derived germ cells, capable of acting as founders of a new generation of mice that consist entirely of cells carrying the carefully designed mutation. In this way, an entire mutant mouse can be resurrected from the culture dish. 8