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DevelopmentW2010.Lecture12notes - Development Bio 120...

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Development Bio 120 Winter 2010 Jeremy Lee Patrick Yuh Eva Murdock Lecture 12 Applications and Medical Implications of Developmental Biology: Reproductive Technologies, Cloning, and Gene Therapies I. Assisted reproductive technology a. In vitro fertilization (Fig. #1.1) 1. Procedure in which eggs and sperm, collected from a male and female, are put together in a petri dish; fertilization occurs, and after several cleavages, the embryo is transferred to the uterus of the female (or another “surrogate” female) to complete development. 2. Basic procedure A. Female is treated with gonadotropins and/or anti-estrogens to stimulate maturation of up to several oocytes. B. Mature oocytes (arrested in meiotic metaphase II) are retrieved from ovaries using an aspiration pipette. C. A sperm sample is collected from the male 1-2 hours prior to fertilization; sperm are "washed"; and most active sperm are combined with oocytes. D. After fertilization and several cleavages, dividing embryos are implanted into the woman's uterus with a catheter. E. In some cases, a small hole is made in the zona pellucida prior to implantation to assist with blastocyst hatching. (Fig. #1.2) F. It is hoped that at least one of the dividing embryos will develop to blastocyst stage, hatch and implant in the uterine wall. In some cases, more than one does and multiple births can result. b. Intracytoplasmic sperm injection (Fig. #2.) In cases in which males have low numbers of viable sperm, this procedure may be used, in which a single sperm is directly injected into the oocyte. c. Gamete intrafollopian transfer (GIFT.) In GIFT, sperm are injected into the fallopian tube at the time of ovulation. This may be done in cases in which cervical or immunological factors are thought to prevent sperm from moving to or up the oviduct, or in cases of unexplained fertility problems. d. Zygote intrafollopian transfer (ZIFT.) In ZIFT or " tubal embryo transfer ," oocytes are fertilized in a dish, but transferred into the fallopian tube rather than into the uterus. II. Reproductive cloning a. The first mammal to be successfully "cloned" was the sheep Dolly in 1997. (Figs. #3, #4) b. Technique to produce Dolly: somatic cell nuclear transfer (SCNT .) 1. Adult somatic cells, in this case mammary epithelial cells, were isolated from a " nucleus donor ", in this case a "White-faced" ewe. 2. Isolated mammary cells were grown in culture. 3. Cells were "pushed" into the G 0 (quiescent) stage of cell cycle. 4. The nucleus was removed from an oocyte from a "Black-faced" ewe (the oocyte donor), creating an enucleated oocyte . 5. Mammary cell from culture was fused with the enucleated egg, using a brief electrical pulse, so that the nucleus would be incorporated into the enucleated oocyte. 6. The oocyte with transferred nucleus was cultured for six days then implanted into the uterus of a foster mother , in this case a "Black-faced" recipient ewe.
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2 7. Development of the implanted oocyte proceeded until Dolly was born. Genetic tests indicated that she was "genetically identical" to the "White-faced" nuclear donor.
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DevelopmentW2010.Lecture12notes - Development Bio 120...

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