Part 2: ODN Modification and Part 4: Fertilization and Development: Difference between pages

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After testing the ODNs, an effective ODN or ODNs are chosen; a rough guideline for an effective ODN is one which depletes the target mRNA to less than 20% of the level of the uninjected at a 10 ng dose, as seen by northern analysis. The ODNs so selected are generally resynthesized in a modified form. The modification suggested by experiments of Baker, et al. (1990) and Dagle, et al. (1990) is used, where 3-4 of the 5'-most and 3'-most phosphodiester bonds are replaced by phosphorothioate bonds, leaving at least 8 unmodified bonds in the center of the ODN (e.g. Kofron et al., 1997).
'''Fertilize embryos'''


Modified oligodeoxynucleotides provide two major advantages over unmodified ODNs. The modified ODNs can deplete the message more effectively than the unmodified ODNs at a much lower concentration (e.g. compare the 2 ng depletion by modified ODN #7 in fig. 4b to the 10 ng unmodified ODN #7 depletion in fig. 4a). Second, the modified ODNs provide a more reliable and reproducible depletion. However, it has been our experience that the phosphorothioate ODNs tend to reach a toxic dose around 5ng. Both the toxicity of the modified ODNs and their ability to further deplete mRNA probably result from the increased stability of phosphorothioate ODNs (Dagle et al., 1990; Woolf et al., 1990). It is also worth noting that we have seen phosphorothioate modification of ODNs cause the ODN to become inactive or less active at 18 degrees Celsius--the temperature at which the oocytes are incubated. In that case, it is appropriate to use the unmodified ODN in order to achieve the best possible depletion--10 ng doses or less of ODN are suggested.
Three hours after the oocytes are transferred to the host frog, the frog is squeezed to lay eggs at 20-30 minute intervals and fertilized with a sperm suspension, as per a normal in vitro fertilization (Holwill et al., 1987). The percentage of experimental oocytes that are laid by the female varies from one experiment to the next, but as long as the female does not stop laying eggs most of the dyed oocytes can be recovered. Of these only a portion of them will fertilize, and a 20-40% rate of fertilization is usual for transferred and uninjected oocytes. Usually a female will give 3-4 good fertilizations per experiment.


Other alternatives to achieve non-toxic depletions also exist. An alternative to phosphorothioate modification which may ameliorate the toxicity of the modified ODNs is to synthesize ODNs with 5' and 3' methoxyethyl phosphoroamidate linkages (Dagle et al., 1990; Heasman et al., 1992); however, we are unaware of any such commercially available ODNs at the time of publication. Another alternative to the use of a high dose phosphorothioate-modified ODN is to utilize two (or theoretically more) ODNs in combination. It has been shown that the combination of oligodeoxynucleotides provides a superior depletion to either one alone, even at a higher dose of ODN (Morgan et al., 1993).
'''Dejelly embryos'''
 
Embryos are dejellied in 2% cysteine pH 7.8 in 0.1X MMR (Appendix A) after the first cell cycle. Briefly, they are gently swirled until the embryos lie close together instead of being held apart by the jelly coat. Then embryos are washed three times in a large excess volume of 0.1X MMR. Both host and transferred embryos are then allowed to develop in 0.1XMMR.
 
As it is easier to distinguish the 5 colors from each other early in development rather than later, the embryos are subdivided into separate dishes at this time and are monitored carefully through development to observe differences between the control and experimental embryos. In addition, embryos may be fixed and frozen for further analysis at later stages.


==Related Articles==
==Related Articles==
*[[Oocyte Transfer Technique]]
*[[Oocyte Transfer Technique]]
*[[Part 1: Choosing Oligodeoxyribonucleotides (ODNs)]]
*[[Part 1: Choosing Oligodeoxyribonucleotides (ODNs)]]
*[[Part 2: ODN Modification]]
*[[Part 3: Host Transfer Technique]]
*[[Part 3: Host Transfer Technique]]
*[[Part 4:Fertilization and Development]]

Revision as of 18:08, 21 December 2009

Fertilize embryos

Three hours after the oocytes are transferred to the host frog, the frog is squeezed to lay eggs at 20-30 minute intervals and fertilized with a sperm suspension, as per a normal in vitro fertilization (Holwill et al., 1987). The percentage of experimental oocytes that are laid by the female varies from one experiment to the next, but as long as the female does not stop laying eggs most of the dyed oocytes can be recovered. Of these only a portion of them will fertilize, and a 20-40% rate of fertilization is usual for transferred and uninjected oocytes. Usually a female will give 3-4 good fertilizations per experiment.

Dejelly embryos

Embryos are dejellied in 2% cysteine pH 7.8 in 0.1X MMR (Appendix A) after the first cell cycle. Briefly, they are gently swirled until the embryos lie close together instead of being held apart by the jelly coat. Then embryos are washed three times in a large excess volume of 0.1X MMR. Both host and transferred embryos are then allowed to develop in 0.1XMMR.

As it is easier to distinguish the 5 colors from each other early in development rather than later, the embryos are subdivided into separate dishes at this time and are monitored carefully through development to observe differences between the control and experimental embryos. In addition, embryos may be fixed and frozen for further analysis at later stages.

Related Articles

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