Part 1: Choosing Oligodeoxyribonucleotides (ODNs): Difference between revisions
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Protocol submitted by Janet Heasman [http://www.xenbase.org/community/person.do?method=display&personId=733&tabId=0] | |||
Choose six to eight ODNs. | Choose six to eight ODNs. | ||
We select ODNs 18 bases in length (based on the experiments of Dagle 1990) complimentary to either the | 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. | Inject ODNs and incubate oocytes. | ||
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 | 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 | Freeze oocytes and perform northern analysis | ||
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==Related Articles== | ==Related Articles== | ||
*[[Oocyte Transfer Technique]] | |||
*[[Part 2: ODN Modification]] | *[[Part 2: ODN Modification]] | ||
*[[Part 3: Host Transfer Technique]] | *[[Part 3: Host Transfer Technique]] | ||
*[[Part 4:Fertilization and Development]] | *[[Part 4:Fertilization and Development]] | ||
Latest revision as of 15:41, 14 January 2010
Protocol submitted by Janet Heasman [1]
Choose six to eight ODNs. 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. 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.