Part 2: ODN Modification
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Protocol submitted by Janet Heasman 
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).
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.
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).