Molecular control of transgene segregation and its escape by a recoverable block of funtion (rbf) system

Abstract

The invention is related to a method and a complex of DNA constructs for providing an increased level of control of transgene segregation in Sexually Reproducing Multicellular Organisms (SRMOs), which are prone to out-crossing with their wild-type or cultivated relatives. The method and constructs allow the farmer to reuse the transgenic crop. without risk. The RBF system comprises one or more Transgenes of Interest (TGIs) encoding desired gene products, one or more Blocking Constructs (BC), and one or more user-controlled means for recovering the blocked functions. The BC has the capacity of blocking at least one function essential for the survival and / or reproduction of the SRMO. Preferably more than one BC flanking the TGIs are used. The BC may be located in close proximity to the TGI, preferably in an intron or flanking the TGIs. The blocked function is recoverable by user-controlled interventions preferably applicable under confined conditions. The intervention is combined with one or more Recovering Constructs (RC). Different types of RBF systems are disclosed. The Double and Triple and Segregating RBF systems and intron-inserted BC are the preferred embodiments of the present invention. The Transgenic Inserts (TI) of the Triple RBF system and their interactions are shown in FIG. 10.

Description

THE TECHNICAL FIELD OF THE INVENTION[0001] The present invention is related to an improved method and complex of DNA constructs, herein called Recoverable Block of Function (RBF) systems, for obtaining an increased security level in the control of transgene segregation and transgene escape including introgression. The method and system also allows farmers to reuse their transgenic crop without risking leakage of transgenes into the environment. The increased security level is achieved by Recoverable Block of Function (RBF) systems comprising one or more blocking nucleotide constructs (BC) closely linked to a Transgene of Interest (TGI), wherein the Blocking Construct (BC) is inserted into an intron of the Transgene of Interest (TGI), if one Blocking Construct (BC) is used or between two Blocking Constructs (BC), if more than one are used, as well as one or more Recovering Constructs (RC), i.e. nucleotide constructs for recovering the blocked function under a user-controlled chemical...

AI Technical Summary

Benefits of technology

[0024] Accordingly, the present invention is related to a method for increasing the security level for controlling transgene segregation and preventing the escape of transgenes into the environment by a molecular control mechanism comprising the steps of constructing one or more complexes of DNA constructs, herein called Recoverable Block of Function (RBF) systems comprising in addition to one or more Transgenes of Interest (TGIs), one or more Blocking Constructs (BCs) located in the close proximity of the Transgene of Interest (TGI), preferably in an intron of a Transgene of Interest (TGI) or if more than one Blocking Constructs (BCs) are used between said Blocking Constructs (BCs), and at least one means for recovering or a recovering tool, i.e. a user-controlled means or intervention for recovering the blocked functions. Said Blocking Construct (BC) has the capacity of blocking at least one function essential for the survival and / or sexual reproduction of a Sexually Reproducing Multicellular Organism (SRMO).

[0026] The presence of a Recovering Construct (RC) in the Recoverable Block of Function (RBF) technology of the present invention enables in the Blocking Construct (BC), the use of transgenes, which can be lethal or cause sterility in the host plant. The use of such genes makes the negative selection required for the effect to take place, absolute and shortens the negative selection time needed as compared to the negative selection in mitigation technology. The security level of Recoverable Block of Function (RBF) systems using Double Blocking Constructs (BCs) is increased because the silencing or mutation in one of the Blocking Constructs (BCs) does not decrease the effect of the second Blocking Construct (BC). Alternatively, the security level of the Recoverable Block of Function (RBF) systems of the present invention can be increased by introducing the Blocking Construct (BC) into an intron of the Transgene of Interest (TGI). This reduces the possibilities that the Blocking Construct (BC) is inactivated by crossing over or mutated without detrimental effects being caused to the Transgene of Interest (TGI), too. Therefore, the combination of the Recoverable Block of Function (RBF) concept with a Multiple, i.e. Double or Triple and / or intron-introduced Blocking Construct (BC) system provides a substantially increased security level in the control of transgene segregation and transgene escape to make the system feasible in transgenic plant and animal DNA technology in practice.

[0028] The transgene is prevented from leaking into the environment through hybridization or out-crossing of the parental transgenic Sexually Reproducing Multicellular Organism (SRMO) with its wild-type relatives or other cultivated non-transgenic or transgenic relatives, by the function of the Blocking Construct (BC) unit, which under natural conditions arrests the essential function of the transgenic Sexually Reproducing Multicellular Organism (SRMO) or any hybrids carrying said Blocking Construct (BC) as long as no user-controlled external intervention is provided. This control of segregation rapidly leads to extinction of the transgene in nature.

[0031] The transgenic Sexually Reproducing Multicellular Organism (SRMO) is prevented from reproducing by withdrawing the user applied intervention, which prevents the action of the Blocking Construct (BC). The action, e.g. out-crossing, arrests and / or alters an essential function effecting the survival, development and / or sexual reproduction of a transgenic Sexually Reproducing Multicellular Organism (SRMO).

[0037] In Multiple Recoverable Block of Function (M-RBF) systems a first Recovering Construct (RC.sub.1) is placed close to a second Blocking Construct (BC.sub.2) in a first Transgenic Insert (TI). Placed in a second non-allelic chromosome a second Recovering Construct-first Blocking Construct (RC.sub.2-BC.sub.1) complex controls the Transgenic Insert (TI) and the first Transgenic Insert (TI) controls the second Transgenic Insert (TI), respectively. The Transgene-of-Interest-Blocking--Constructs (BC.sub.1-TG.sub.1-BC.sub.2) complex controls another Transgene-of-Interest-Blocking-Construct (TGI-BC) complex with increased reliability and negative selection of Transgene of Interest (TGI).

Problems solved by technology

Risk groups are found among certain crop species, for example corn, certain oil producing plants such as Brassicae, but also among trees, which are a specific problem due to their long life span and great production of pollen.

Even if the problem at present is mainly restricted to plants, it is likely that similar problems will be encountered when animals are becoming more serious targets in the transgenic production methods.

While the risks to human or animal health of a particular transgene and its product can be tested and measured, the impact of gene escape is more complex to assess.

However, male sterility does not prevent escape of the transgene into the environment, because the pollinated female (MS) plants are still capable of producing hybrid seeds, which if shattering may, stay behind in the field after harvesting.

Blocking of the expression of this gene leads to an unrecoverable blocking of fertilization.

The fundamental problem with said technique is that once the plants have been tetracycline treated, i.e. the killer gene has been activated, they cannot be rescued.

Said so called "terminator technology" has encountered negative public attention because it gives the seed producing companies the possibility to control the market of transgenic seed production.

If the repressor gene is not activated, the disrupter gene destroys the cell and the functions of the plant.

The TM genes are positive or neutral to the crop but deleterious to the weed.

However, the mitigation genes can not entirely prevent transgene escape, because they are neither lethal nor even adverse for crop plants.

Accordingly, transgenetic mitigation technology does not prevent the crossing between different lines of the same crops.

The effect of mitigation genes is generally achieved only after prolonged negative selection periods during which time possible silencing or mutations in the mitigating genes reduce their effect and consequently act against achieving the prevention of transgene escape.

Tandems of mitigation genes do not solve the problem of crossing between different lines of related crop plants.

Most of the systems intended for providing control of escape of transgenic plants have been described in prior art are unrecoverable.

In other words, they cannot be reused once the recovery system has been applied.

A further limitation in some of the above described systems is that they require vegetative propagation, because fertilization or embryo development is arrested.

Accordingly, the control situation is far from ideal in spite of the multitude of different approaches suggested in prior art.

When starting from the use of mitigating genes, it can be concluded that sufficient security is not achieved with mitigation genes.

Prior art systems applying lethal or sterility causing genes have been described and used, but sufficient security levels cannot be ensured, especially when longer selection times are required.

Furthermore, some of the prior art systems applying lethal or sterility causing genes require undesired steps, such as distribution of chemicals into the environment.

This is not well seen by the users or public at large.

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