Part 1: Choosing Oligodeoxyribonucleotides (ODNs): Difference between revisions

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This chapter describes a method that has been used successfully to study the roles of a number of maternal mRNAs in Xenopus embryos (Kofron et al., 1997; Heasman et al., 1992, 1994; Weeks et al. 1991). The aim of the technique is to study the role of these genes by creating a milieu in which the gene is not fully active. This is achieved by reducing the maternal pool of RNA for the gene of interest and studying the effect that depletion has on development. Depletion of the maternal mRNA of interest is accomplished by injection of antisense oligodeoxyribonucleotides (ODNs) and subsequent fertilization of these oocytes in order to study the effects of depletion. While these underexpression experiments are technically more demanding than overexpression experiments, they are more rewarding in that they can reveal directly the functions of the protein product of the mRNA of interest.
Protocol submitted by Janet Heasman [http://www.xenbase.org/community/person.do?method=display&personId=733&tabId=0]


Underexpression experiments require injection of antisense ODNs complementary to the target mRNA into fully grown ovarian oocytes. Oocytes contain an endogenous RNAse H activity that cleaves RNA/DNA duplexes, therefore mRNA bound to the ODNs is cleaved by RNAse H and then broken down further by other nucleases (Dash et al., 1987). A single injection of ODN at a single time point effectively reduces maternal RNA levels, as from the time the oocyte is fully grown until the embryo reaches the 4000-cell stage no new RNA is synthesized. Therefore new mRNA is not produced until synthesis of RNA from the embryonic genome begins (zygotic synthesis), and zygotic synthesis of the mRNA of interest may not begin until well after the 4000-cell stage. Thus, injection of ODN into the oocyte effectively removes mRNA until the 4000-cell stage. However, the continual synthesis of zygotic mRNA makes antisense depletion a poor choice for targeting zygotic mRNAs, as ODNs would need to be added continually to prevent depleted mRNA being replaced. This portion of the powerful host transfer, or underexpression, technique--the depletion of RNA from the oocyte--is adapted from the original studies in Xenopus oocytes by the laboratories of Walder, Coleman, and Weeks (see Dagle et al., 1990, 1991; Weeks et al., 1991; and Shuttleworth et al., 1988a,b).
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.


The second part of the underexpression experiment involves the fertilization of oocytes taken from one frog and then transferred into a different frog, using a method devised by Subkelny, et. al. (1961) and Brun, et.al. (1975) and refined in this lab (Holwill et.al., 1987). Although it would seem simpler to bypass the process of removing ovary from one frog only to place it into another, we do not inject oligodeoxyribonucleotides directly into fertilized eggs. Briefly, ODNs are more toxic in eggs, are not as effective in degrading mRNAs and may be inherited in a mosaic fashion, making results difficult to interpret (Heasman et.al., 1992; and Woolf et.al., 1990).
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.


Underexpression has been utilized in this laboratory to study the functions of cytoskeletal proteins, adhesion proteins, signaling molecules and transcription factors in Xenopus embryos. In some cases specific functions of the proteins were revealed, while for other molecules, no phenotype was seen when the mRNA was depleted (Kofron et.al., 1997; Heasman et.al., 1992, 1994; data not shown).
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.


Unfortunately, the disadvantage of the underexpression technique is that in cases where no phenotype results, it is extremely difficult to determine whether the lack of effect is due to an insufficient depletion of the mRNA or protein (e.g. if the protein has a very long half-life), or whether the molecule is redundant in function. For this reason, it is important to analyze the level of gene function as accurately as possible. Therefore when an assay exists to test gene activity (e.g. a kinase assay for a maternal kinase) it should be used to determine the level of gene function after underexpression. Also, when antibodies are available Western analysis and/or immunostaining on ODN-injected oocytes should be utilized to show that depletion of the mRNA does lead to a reduction in the level of protein. This reduction would be expected to be greatest at the late blastula stage, when the maternal pool of protein will be most exhausted and before zygotic synthesis of RNA is underway .Underexpression is a powerful tool for analysis of gene function in Xenopus laevis, but it does have limitations.
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]]

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.

Related Articles

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