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.

1 . A method for providing an increased security level for controlling transgene segregation in a Sexually Reproducing Multicellular Organism (SRMO) and/or for providing automatic prevention of transgene escape, characterized in that the method comprises a molecular mechanism including the steps of 
 (a) constructing one or more Recoverable Block of Function (RBF) systems or complexes of DNA constructs for inserting into a recipient cell or cell-line in addition to a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs) encoding one or more desired gene products, one or more Blocking Constructs (BC), and providing at least one user-controlled means for recovering the blocked functions; 
 (i) said Blocking Construct (BC) having the capacity of blocking at least one molecular or physiological function essential for the survival or reproduction of the Sexually Reproducing Multicellular Organism (SRMO), which Blocking Construct (BC) is located in the same chromosome as, and in the close proximity of the Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs) preferably in an intron of a Transgene of Interest (TGI) or flanking each side of a Transgenic Insert (TI) comprising;  
 (ii) said means for recovering the blocked function comprising one or more user-controlled intervention optionally combined with one or more Recovering Constructs (RC), which are placed close to a Blocking Construct (BC) in a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs) and/or in non-allelic chromosomes; and  
   (b) recovering the blocked function in order to allow normal development during cultivation and reproduction of said Sexually Reproducing Multicellular Organism (SRMO) for production purposes by applying, at a susceptible stage of the developmental or reproductive cycle of said Sexually Reproducing Multicellular Organism (SRMO), one or more user-controlled interventions;    with the proviso that when solely one Blocking Construct is used, said Blocking Construct (BC) or a part of it is placed in one intron of the only Transgene of Interest (tgi).    
     
     
         2 . The method according to  claim 1 , characterized in that the Blocking Constructs (BCs) are flanking each side of a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs), when two or more Blocking Constructs (BCs) are used.  
     
     
         3 . The method according to  claim 1 , characterized in that the Recoverable Block of Function (RBF) system is a Double Recoverable Block of Function (D-RBF) system comprising at least two Blocking Constructs (BCs), which are the same or different and which are recoverable by the same or different recovering mechanisms, flanking each side of the Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs).  
     
     
         4 . The method according to  claim 1 , characterized in that a the Recoverable Block of Function (RBF) system is a Multiple, preferably a Triple Recoverable Block of Function (M-RBF, T-RBF) system comprising two, preferably three or more Transgenic Inserts (TIs) placed in three or more non-allelic chromosomes, in which the first Transgenic Insert (TI) comprises at least two different Blocking Constructs (BCs), the second Transgenic Insert (TI) comprises one or more first Recovering Constructs (RCs) linked to an optional Blocking Construct (BC) being the same or different from one of the Blocking Constructs (BCs) of the first Transgenic Insert (TI) and a third Transgenic Insert (TI) comprising one or more second Recovering Constructs (RCs) which are different from the first Recovering Constructs (RCs) and which are linked to a Blocking Construct (BC) corresponding to one of the first Transgenic Insert (TI) which is not the same as the optional Blocking Construct (BC) of the second Transgenic Insert (TI).  
     
     
         5 . The method according to  claim 1 , characterized in that for obtaining a Multiple Recoverable Block of Function (M-RBF) system the recipient transgenic Sexually Reproducing Multicellular Organism (SRMO) is provided with two Transgenic Inserts (TIs) of a Reversed Segregating Recoverable Block of Function (RS-RBF) system in two non-allelic chromosomes without the Transgene of Interest (TGI) and a transformation vector comprising two Blocking Constructs (BCs) placed on each side of a cloning site for desired Transgenes of Interest (TGIs) and implying said vector to transform the recipient transgenic Sexually Reproducing Multicellular Organism (SRMO) line into a third non-allelic chromosome.  
     
     
         6 . The method according to  claim 1 , characterized in that the Recoverable Block of Function (RBF) system is a Segregating Recoverable Block of Function (S-RBF) system comprising a Recovering Construct (RC) and a Blocking Construct (BC) placed in different non-allelic chromosomes, with the Blocking Construct. (BC) in an intron of a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs).  
     
     
         7 . The method according to  claim 1 , characterized in that an Induced Recoverable Block of Function (I-RBF) system comprises a Recovering Construct (RC) placed in the same chromosome as the Blocking Construct (BC) but expressed under a different promoter, with the Blocking Construct (BC) or part of it placed in an intron of a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs).  
     
     
         8 . The method according to  claim 1 , characterized in that at least one nucleotide sequence in the Blocking Construct (BC) the expression of which blocks in a constitutive, organ-specific or spatiotemporal manner a molecular or physiological function essential for the survival or reproduction of a transgenic Sexually Reproducing Multicellular Organism (SRMO).  
     
     
         9 . The method according to  claim 1 , characterized in that the removal of the effect of the Blocking Construct (BC) is achieved by applying a user-controlled intervention, which at a susceptible stage in the development and/or reproductive cycle of the transgenic Sexually Reproducing Multicellular Organism (SRMO) upheaves the constitutive, organ-specifically or spatiotemporally achieved morphological or physiological block preventing hybridization and/or sexual reproduction of the transgenic Sexually Reproducing Multicellular Organism (SRMO).  
     
     
         10 . The method according to  claim 9 , characterized in that the susceptible stage essential for the survival and/or reproduction is selected from a group consisting of early or late seed germination, flowering, formation of dwarf phenotype, incapability of forming inflorescence flowers and/or fruits, incapability of germination, vegetation, rooting and photosynthesis.  
     
     
         11 . The method according to  claim 9 , characterized in that the susceptible stage essential for the survival and/or reproduction is selected from a group consisting of early or late seed germination.  
     
     
         12 . The method according to  claim 1 , characterized in that the removal of the effect of the blocked function comprises a user-controlled externally or internally regulatable molecular mechanism optionally applicable under confined conditions.  
     
     
         13 . The method according to  claim 12 , characterized in that the user-controlled, externally and/or internally regulatable molecular mechanism for removal of the blocked effect is selected from a group consisting of 
 (a) adding at least one externally applicable substance;    (b) applying at least one external chemical or physical stimulus capable of activating the optional Recovering Construct (RC);    (c) repressing the promoter of the Blocking Construct (BC);    (d) silencing the mRNA expression of the nucleotide sequence of the Blocking Construct (BC) by antisense RNA technology;    (e) allowing the expression of a protein capable of inactivating the protein, which is encoded by the nucleotide sequence of the Blocking Construct (BC) by applying an externally activatable molecular mechanisms;    (f) allowing the expression of an enzyme producing a substance capable of inactivating the protein expressed by a nucleotide sequence of the Blocking Construct (BC);    (g) allowing the expression of an enzyme capable of inactivating a toxic substance produced by the enzyme;    (h) allowing the expression of an enzyme capable of producing a substance compensating the deficiency caused by action of the Blocking Construct (BC);    (i) allowing the expression of an enzyme capable of producing a substance altering the action caused by the Blocking Construct (BC);    (j) allowing specific nucleic acid and/or protein binding;    (k) applying proteolysis;    (l) allowing protein-protein interaction;    (m) expressing an enzyme capable of detoxifying a toxic substance one;    (n) expressing a substance capable of compensating or inactivating the toxic overproduced substance    (o) expressing a substance capable of interacting with a toxic substance;    (p) providing an enzyme capable of converting a toxic or overproduced substance with wrong spatiotemporal specificity;    (q) compensating externally the deficiency caused by the Blocking Construct (BC);    (r) inducing externally a responsive recovering DNA, RNA, protein or metabolite;    (s) expressing constitutively the nucleotide sequence of the Recovering Construct (RC);    (t) expressing spatiotemporally the nucleotide sequence of the Recovering Construct (RC); and    (u) allowing intraline crossing when the Recoverable Block of Function (RBF) system is a Segregating Double or a Multiple Recoverable Block of Function (RBF) system.    
     
     
         14 . The method according to  claim 1 , characterized in that the user-controlled, externally regulatable molecular mechanism for recovering the blocked function comprises allowing intraline crossing supporting homozygous conditions.  
     
     
         15 . A complex of DNA constructs or a Recoverable Block of Function (RBF) system for providing an increased security level in the control transgene segregation in a Sexually Reproducing Multicellular Organism (SRMO) and/or for providing an automatic prevention of introgression, characterized in that said complex of DNA constructs comprises 
 (a) a Transgenic Insert (TI) comprising at least one Transgene of Interest (TGI) encoding one or more desired gene products;    (b) one or more Blocking Constructs (BC), which are recoverable by user-controlled means for recovering, said Blocking Construct (BC) including at least one nucleotide sequence having the capacity of blocking at least one molecular or physiological function essential for the survival or reproduction of the Sexually Reproducing Multicellular Organism (SRMO) and being located in the same chromosome as and in the close proximity of the Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs) preferably in an intron of a Transgene of Interest (TGI) or flanking each side of a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs);    (c) one or more optional Recovering Constructs (RC) which are placed close to one of the Blocking Constructs (BCs) in an Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs) placed in one or more non-allelic chromosomes;    with the proviso that when solely one Blocking Construct (BC) is used, said Blocking Construct (BC) or part of it is placed in an intron of the only Transgene of Interest (TGI).    
     
     
         16 . The complex of DNA constructs according to  claim 15 , characterized in that the Blocking Constructs (BCs) are flanking on each side of the Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs), when two or more Blocking Constructs (BCs) are used forming a self-controlling and self-supporting entity.  
     
     
         17 . The complex of DNA constructs according to  claim 15 , characterized in that the Recoverable Block of Function (RBF) system is a Double Recoverable Block of Function (D-RBF) system comprising Blocking Constructs (BCs), which are at least two and the same or different and situated at both sides of a Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs) and recoverable by the same or different recovering mechanisms.  
     
     
         18 . The complex of DNA constructs according to  claim 15 , characterized in that the Recoverable Block of Function (RBF) system is a Multiple Recoverable Block of Function (M-RBF) system comprising two, preferably three or more Transgenic Inserts (TIs) placed in three or more non-allelic chromosomes, in which the first Transgenic Insert (TI) comprises at least two different Blocking Constructs (BCs), the second Transgenic Insert (TI) comprises one or more first Recovering Constructs (RCs) linked to an optional Blocking Construct (BC) corresponding to one of the Blocking Constructs (BCs) of the first Transgenic Insert (TI) and the third Transgenic Insert (TI) comprising one or more second Recovering Constructs (RCs) which are the same or different from the first Recovering Constructs (RCs) and which are linked to a Blocking Construct (BC) being the same as one of the first Transgenic Insert (TI) which is not the same as the optional Blocking Construct (BC) of the second Transgenic Insert (TI).  
     
     
         19 . The complex of DNA constructs according to  claim 15 , characterized in that in a Multiple Recoverable Block of Function (M-RBF) system the recipient transgenic Sexually Reproducing Multicellular Organism (SRMO) is provided with two Transgenic Inserts (TIs) of a Reversed Segregating Recoverable Block of Function (RS-RBF) system in two non-allelic chromosomes without the Transgene of Interest (TGI) and a transformation vector comprising to Blocking Constructs (BCs) placed on each side of a cloning site for desired Transgenes of Interest (TGIs) and implying said vector to transform the recipient transgenic Sexually Reproducing Multicellular Organism (SRMO) line into a third non-allelic chromosome.  
     
     
         20 . The complex of DNA constructs according to  claim 15 , characterized in that the Recoverable Block of Function (RBF) system is an Induced Recoverable Block of Function (1-RBF) system comprising a Recovering Construct (RC) placed in the same chromosome as the Blocking Construct (BC) with the Blocking Construct (BC) in an intron of the Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs).  
     
     
         21 . The complex of DNA constructs according to  claim 15 , characterized in that the Recoverable Block of Function (RBF) system is a Segregating Recoverable Block of Function (S-RBF) system comprising a Recovering Construct (RC) and a Blocking Construct (BC) placed in different non-allelic chromosomes with the Blocking Construct (BC) or part of it placed in an intron of the Transgenic Insert (TI) comprising one or more Transgenes of Interest (TGIs).  
     
     
         22 . The complex of DNA constructs according to  claim 15 , characterized in that an inducible Recovering Construct (RC) is activatable in response to a user-controlled intervention, which recovers the function essential for the survival and sexual reproduction of transgenic Sexually Reproducing Multicellular Organism (SRMO) and which function is blocked by a Blocking Construct (BC) expressed under the same or a different type of promoter.  
     
     
         23 . The complex of DNA constructs according to  claim 15 , characterized in that the two nucleotide sequences of the Blocking Construct (BC) forming a Double Recoverable Block of Function (D-RBF) system are recoverable by the same or different means for recovering.  
     
     
         24 . The complex of DNA constructs according to  claim 15 , characterized in that an inducible Recovering Construct (RC) is activatable in response to a user-controlled intervention, which recovers the function blocked by the Blocking Construct (BC) expressed under the same or a different promoter.  
     
     
         25 . The complex of DNA constructs according to  claim 15 , characterized in that the Blocking Construct (BC) comprises at least one nucleotide sequence, the constitutive, organ specific or spatiotemporal expression of which blocks an essential molecular or physiological function at DNA, mRNA, protein or metabolite level and/or causes a phenotypical, physiological or morphological alteration preventing free hybridization and/or reproduction of an out-crossing, transgenic Sexually Reproducing Multicellular Organism (SRMO) under natural, uncontrolled conditions leading to its extinction.  
     
     
         26 . The complex of DNA constructs according to  claim 15 , characterized in that the function blocked by the Blocking Construct (BC) of the Recoverable Block of Function (RBF) system, is recovered by applying a user-controlled externally or internally regulatable molecular mechanism optionally applicable under confined conditions.  
     
     
         27 . The complex of DNA constructs according to  claim 26 , characterized in that the user-controlled intervention for recovering the blocked function is selected from a group consisting of 
 (a) adding at least one externally applicable substance;    (b) applying at least one external chemical or physical stimulus capable of activating the optional Recovering Construct (RC);    (c) repressing the promoter of the Blocking Construct (BC);    (d) silencing the blocking gene mRNA expression by antisense RNA technique;    (e) allowing the expression of a protein capable of inactivating the Blocking Construct (BC) encoding protein by an externally activatable molecular mechanisms;    (f) allowing the expression of an enzyme capable of producing a substance inactivating the protein expressed by the Blocking Construct (BC);    (g) allowing the expression of an enzyme capable of inactivating a toxic substance produced by the enzyme;    (h) allowing the expression of an enzyme capable of producing a substance compensating the deficiency caused by the action of the Blocking Construct (BC).    (i) allowing the expression of an enzyme capable of producing a substance compensating the alteration caused by the action of the Blocking Construct (BC).    (j) allowing specific nucleic acid and/or protein binding;    (k) applying proteolysis;    (l) allowing protein-protein interaction;    (m) expressing an enzyme capable of detoxifying a toxic substance one;    (n) expressing a substance capable of compensating or inactivating the toxic overproduced substance    (o) expressing a substance capable of interacting with a toxic substance;    (p) providing an enzyme producing a toxic or overproduced substance;    (q) compensating externally the deficiency caused by the Blocking Construct (BC);    (r) inducing externally a responsive recovering DNA, RNA, protein or metabolite;    (s) expressing constitutively the nucleotide sequence of the Recovering Construct (RC);    (t) expressing spatiotemporally the nucleotide sequence of the Recovering Construct (RC); and    (u) allowing intraline crossing when the Recoverable Block of Function (RBF) system is a multiple Recoverable Block of Function (RBF) system.    
     
     
         28 . The complex of DNA constructs according to  claim 15 , characterized in that the user-controlled externally regulatable molecular mechanism for recovering the blocked function comprises allowing intraline crossing.  
     
     
         29 . The complex of DNA constructs according to  claim 15 , characterized in that the Blocking Construct (BC) comprises a nucleotide sequence expressing in the host organism in a constitutive, organ-specific or spatiotemporal manner and the Recovering Construct (RC), comprises a nucleotide sequence capable of being recovered by a user-controlled intervention regulating a treatment-responsive nucleotide sequence or its gene product or by constitutive expression of the nucleotide sequence in the Recovering Construct (RC) and/or its gene product.  
     
     
         30 . The complex of DNA constructs according to  claim 29 , characterized in that the Blocking Construct (BC) comprises a nucleotide sequence which expresses an enzyme in an organ-specific, developmental or spatiotemporal manner, an antisense mRNA of an enzyme essential for the survival or sexual reproduction of the host organism.  
     
     
         31 . The complex of DNA constructs according to  claim 30 , characterized in that the Blocking Construct (BC) comprises a synthetic nucleotide sequence adapted to host organism preference and/or to intron preference of the Transgene of Interest (TGI) encoding the desired product in a host organism.  
     
     
         32 . The complex of DNA constructs according to  claim 31 , characterized in that the synthetic nucleotide sequence encodes the enzyme barnase, having the SEQ ID NO:1:, and has a nucleotide sequence adapted to plant preference and/or adapted to an intron of a plant gene said sequence being SEQ ID NO:3:.  
     
     
         33 . The complex of DNA constructs according to  claim 15 , characterized in that the Recovering Construct (RC) comprises a repressor peptide/protein binding the operon sequence, which controls the expression of the blocking nucleotide sequence.  
     
     
         34 . The complex of DNA constructs according to  claim 33 , characterized in that the repressor peptide/protein is a Tn10 tet repressor protein and the operon sequence is a tet-operator DNA sequence.  
     
     
         35 . The complex of DNA constructs according to  claim 15 , characterized in that the Recovering Construct (RC) comprises a nucleotide sequence expressing an antisense RNA of the blocking nucleotide sequence and being capable of activating a silencing mechanism of the Blocking Construct (BC) expression compensating the deficiency resulted by the action of the Blocking Construct (BC).  
     
     
         36 . The complex of DNA constructs according to  claim 15 , characterized in that the Recovering Construct (RC) comprises a nucleotide sequence expressing a protein inactivating the protein expressed by Blocking Construct (BC).  
     
     
         37 . The complex of DNA constructs according to  claim 36 , characterized in that the nucleotide sequence of the Recovering Construct (RC) expresses Barstar protein inactivating the Barnase nuclease by binding to it.  
     
     
         38 . The complex of DNA constructs according to  claim 15 , characterized in that the Blocking Construct (BC) comprises a synthetic nucleotide sequence adapted to be inserted into the intron of a Transgene of Interest (TGI) by providing intron nucleotide sequence preference.  
     
     
         39 . The complex of DNA constructs according to  claim 38 , characterized in that the synthetic nucleotide sequence is adapted for insertion is a nucleotide sequence substantially identical to the barnase gene sequence and having the SEQ ID NO:3:.  
     
     
         40 . The complex of DNA constructs according to  claim 38 , characterized in that the synthetic sequences of barnase and barstar genes are adapted for plant expression comprise SEQ ID NO:1: and SEQ ID NO:2:, respectively.  
     
     
         41 . The complex of DNA constructs according to  claim 38 , characterized in having a synthetic polycloning site (SEQ ID NO:4:) designed with an Open Reading Frame (ORF) and adapted for β-galactosidase expression in a cloning vector.  
     
     
         42 . The complex of DNA constructs according to  claim 15 , characterized in that the increased security level means that the frequency of inactivation of the Blocking Construct (BC) is lower than 10 −6 , preferably lower than 10 −8 , more preferably lower than 10 −10 , most preferably lower than 10 −12.    
     
     
         43 . The complex of DNA constructs according to  claim 15 , characterized in that the increased security level means that a 100% prevention of transgenic escape is achieved.  
     
     
         44 . A cloning vector comprising one or more of the Blocking Constructs (BCs) useful for assembling the complex of DNA constructs according to  claim 15 , characterized in that the vector is provided with a polycloning site for convenient transfer of the Transgene of Interest (TGI) into the appropriate position in a chromosome of a Sexually Reproducing Multicellular Organism (SRMO).  
     
     
         45 . The cloning vector according to  claim 44  characterized in that the polycloning site is (SEQ ID NO:4:).  
     
     
         46 . Cells or cell-lines of a Sexually Reproducing Multicellular Organism (SRMO) characterized in that they comprise one or more of the Transgenic Inserts (TIs) for assembling the complex of DNA constructs according to  claim 15  for convenient complementation with the vector provided with the Transgenes of Interest (TGIs) comprising one or more Transgenes of Interest (TGIs) in a cloning vector(s) according to claims  44 - 45 .  
     
     
         47 . A transgenic plant characterized in that it comprises one or more of the Transgenic Inserts (TIs) selected from a group consisting of one or more Blocking Constructs (BCs) optionally combined with one or more Recovering Constructs (RCs) with at least one Transgenic Insert (TI) having one or more Transgenes of Interest (TGIs) or a cloning site for inserting one or more Transgenes of Interest (TGIs) for assembling the complex DNA constructs or Recoverable Block of Function (RBF) system according to  claim 15 .  
     
     
         48 . A transgenic non-human Sexually Reproducing Multicellular Organism (SRMO) characterized in that it comprises one or more Transgenic Insert (TI) selected from a group consisting of one or more Blocking Constructs (BCs) optionally combined with one or more Recovering Constructs (RCs) with at least one Transgenic Insert (TI) having one or more Transgenes of Interest (TGIs) or a cloning site for inserting one or more Transgenes of Interest (TGIs) for assembling the complex of DNA constructs according to  claim 15.