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By Andrew Ward

Univ. of tub, united kingdom. Brings jointly the fundamental molecular, genetic, and embryological equipment utilized in trendy laboratories for the id and research of imprinted genes. each one approach is defined intimately. define layout.

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4. After 3 min in the ethanol solution, transfer oocytes to 2 mL of standard medium M2 to wash out ethanol. 5. Transfer oocytes to culture drops of medium CZB (18) containing 5 µg/mL cytochalasin B or 1 µg/mL cytochalasin D. Culture for 4 h to inhibit extrusion of the second polar body, then transfer oocytes to standard medium CZB and culture for another 4 h. 6. Eight to 12 h after ethanol treatment, select 1-cell ova which have two pronuclei and no polar body. This should be done with a microscope under phase contrast or differential interference contrast optics.

Oocytes of hybrid females, such as (C57BL female × C3H, CBA, or SLJ male)F1 are the best for parthenogenetic activation. For inbred strains, C57BL are good, 36 Mann while 129/Sv are poor. Usually the problems lie not in activation but in the viability of the ovum following the experimental procedure. A potentially gentler method than ethanol treatment for activating oocytes is to culture them for 8 h in medium CZB containing 10 mM strontium chloride instead of calcium chloride. Again, 5 µg/mL cytochalasin B or 1 µg/mL cytochalasin D is used to inhibit polar body extrusion (32).

B) Blastocyst outgrowth 4 d after transfer to feeder plate. Note layer of trophectoderm cells surrounding outgrowth. (C) Picked outgrowth in trypsin-EDTA, (D) disaggregated outgrowth, (E) two 129/SvImJ ES cell colonies at passage 1 after 3 d of culture; note patch of trophectoderm cells at top right. These colonies were derived from a 4-well passaged in its entirety into a 12-well, and (F) colonies in (E) after 4 d of culture. All objects at 100× (original magnification) and under phase-contrast optics.

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