Part 1: Choosing Oligodeoxyribonucleotides (ODNs) and Part 2: ODN Modification: Difference between pages

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Protocol submitted by Janet Heasman [http://www.xenbase.org/community/person.do?method=display&personId=733&tabId=0]
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 5Õ-most and 3Õ-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).


Choose six to eight ODNs.
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.
We select ODNs 18 bases in length (based on the experiments of Dagle 1990) complimentary to either the 5' untranslated region (UTR), the 3' UTR, or the coding region of the sequence of the mRNA. Parts of the sequence are chosen that do not have the same base occurring three or more times consecutively and which do have a balance of purines and pyrimidines. Utilizing these restrictions, six to eight ODNs are chosen at random, synthesized and desalted (Biosynthesis, Inc.). Subsequently the ODNs are resuspended in sterile (0.2 um) filtered distilled water at a concentration of 1mg/ml and stored in 10 µl aliquots in a -80 freezer until just before use.


Inject ODNs and incubate oocytes.
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 5Õ and 3Õ 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).
Small numbers of full-grown stage 6 oocytes are then manually defolliculated from pieces of ovary in oocyte culture medium (OCM, Appendix A, for further details see 'Defolliculate and inject' under 'The host transfer technique') and stored at 18 degrees for use. The six test ODNs are spun on an eppendorf centrifuge at 20,000 rpm for 10 mins at 4 degrees Celsius and placed on ice. Each ODN is injected into approximately 5 oocytes per dose in doses of 5 and 10 ng. Injections are accomplished using glass needles pulled in a moving coil microelectrode puller (model 753, Campden Instruments Ltd., Genetic Research Instrumentation Ltd.). The glass needle is broken off at the very tip and the needle is calibrated on a high pressure injection system (Medical Systems, Inc. PLI-100) by collecting the volume of 10-1 second injections into a 1 µl capillary ('microcaps,' disposable 1ul micropipettes, Drummond). Only needles which conform to a volume of 2-10 nl per 1 second injections are used. Needles are attached to a micromanipulator (Leitz) and oocytes are then injected in the equatorial zones while in OCM under a dissecting microscope (Leica Wild M8) and incubated overnight to deplete the mRNA thoroughly.
 
Freeze oocytes and perform northern analysis
The next day injected oocytes are frozen for northern blot analysis along with uninjected controls. Northern blot analysis is carried out utilizing procedures described in Kofron, et. al. (1997), isolating RNA from 2-5 oocytes and loading 1-2.5 oocyte equivalents per lane (depending on the abundance of the mRNA of interest). Fig 4(a) is an example of such a northern blot in which 7 ODNs are tested in this fashion to determine which deplete plakoglobin mRNA.
 
It is interesting that, although all six ODNs are completely complementary to the target mRNA not all of them are effective in depleting the RNA (fig. 4a). This represents a common result for depletion of a message by a number of ODNs, and presumably reflects the fact that as a result of folding or protein binding, only parts of the mRNA are available for the annealing of ODNs.
 
==Related Articles==
*[[Oocyte Transfer Technique]]
*[[Part 2: ODN Modification]]
*[[Part 3: Host Transfer Technique]]
*[[Part 4:Fertilization and Development]]

Revision as of 18:04, 21 December 2009

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 5Õ-most and 3Õ-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 5Õ and 3Õ 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).