Method for producing dna vectors from molecular bricks containing sequences of interest
AssigneesUniversite Des Sciences Et Technologies De Lille 1, Centre National De La Recherche Scientifique
InventorsGeoffroy DE BETTIGNIES, Carine DE BETTIGNIES, Sylvain JULIEN
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Abstract
Disclosed is a method for producing, in one step, “made to measure” double-stranded DNA vectors from molecular bricks including sequences of interest in the presence of a one and only type IIs restriction enzyme.
Claims22
A method for producing a circular double-stranded DNA vector comprising at least two sequences of interest, said method comprising:
- a) a step of simultaneously contacting at least two molecular building blocks, which are different from one another, in the presence of a single restriction enzyme, said single restriction enzyme being a type IIs restriction enzyme,
- each molecular building block being a linear double-stranded DNA molecule and containing:
- (i) a sequence of interest with no specific recognition site of the aforementioned type IIs restriction enzyme and comprising at least one unit, said unit being a functional unit or a non-functional unit, said unit comprising at least one module, said module being a functional module or a non-functional module,
- (ii) two double-stranded DNA adapters, flanked upstream and downstream of said sequence of interest, each double-stranded DNA adapter consisting of a sequence of at least 12 nucleotides, which sequence contains:
- a single and only recognition site of the aforementioned type IIs restriction enzyme,
- the recognition site of the aforementioned type IIs restriction enzyme of the adapter upstream of said sequence of interest and the recognition site of the aforementioned type IIs restriction enzyme of the adapter downstream of said specific sequence being convergent,
- which step leads:
- to the elimination by cleaving of the recognition sites of the type IIs restriction enzyme used,
- to the formation of a cohesive single-stranded suture of at least 2 nucleotides at each of the ends of said sequence of interest,
- said cohesive single-stranded suture of at least 2 nucleotides upstream of one of the at least two sequences of interest being complementary to said cohesive single-stranded suture of at least 2 nucleotides downstream of another sequence of interest,
- to the pairing by nucleotide complementarity of the aforementioned cohesive single-stranded sutures of at least 2 nucleotides, and
- to the positioning of the sequences of interest contiguously with one another in an order and a single and defined direction,
- b) a step of ligation of the aforementioned cohesive single-stranded sutures of at least 2 nucleotides,
- so as to obtain a circular double-stranded DNA vector.
A double-stranded DNA vector with no multiple cloning site and allowing the simultaneous expression of multiple transgenes and consisting of a sequence comprising the following functional units:
- U1, nxU2a and mxU2b,
- U1 representing a bacterial functional unit,
- U2a representing an expression functional unit of which the promoter is dependent on RNA polymerase II and of which the expression product is a protein,
- U2b representing an expression functional unit of which the promoter is dependent on RNA polymerase III and of which the expression product is a non-coding RNA,
- n being greater than or equal to 0,
- m being greater than or equal to 0, on the condition that n+m≥2,
- the unit U1 possibly being contiguous with the units nxU2a, these in turn possibly being contiguous with the units mxU2b,
- the unit U1 preferably being contiguous with the units nxU2a, these in turn preferably being contiguous with the units mxU2b.
A double-stranded DNA vector with no multiple cloning site and allowing selection of the integration of transgenes by non-homologous recombination in the target genome and consisting of a sequence comprising the following functional units:
- U1, nxU2a, mxU2b and U3a,
- U1 representing a bacterial functional unit,
- U2a representing an expression functional unit of which the promoter is dependent on RNA polymerase II and of which the expression product is a protein,
- U2b representing an expression functional unit of which the promoter is dependent on RNA polymerase III and of which the expression product is a non-coding RNA,
- n being greater than or equal to 0,
- m being greater than or equal to 0, on the condition that n+m≥2,
- U3a representing a positive selection cassette,
- the unit U1 possibly being contiguous with the units nxU2a, these in turn possibly being contiguous with the units mxU2b, these in turn possibly being contiguous with U3a,
- the unit U1 preferably being contiguous with the units nxU2a, these in turn preferably being contiguous with the units mxU2b, these in turn preferably being contiguous with U3a.
A double-stranded DNA vector with no multiple cloning site and allowing selection of the simultaneous integration of multiple transgenes by non-homologous recombination in the target genome and consisting of a sequence comprising the following functional units:
- U1, U3b, nxU2a, mxU2b, U3a and U3c,
- U1 representing a bacterial functional unit,
- U3b representing motif 5′ of a homologous recombination sequence X
- U2a representing an expression functional unit of which the promoter is dependent on RNA polymerase II and of which the expression product is a protein,
- U2b representing an expression functional unit of which the promoter is dependent on RNA polymerase III and of which the expression product is a non-coding RNA,
- n being greater than or equal to 0,
- m being greater than or equal to 0, on the condition that n+m≥2,
- U3a representing a positive selection cassette,
- U3c representing motif 3′ of a homologous recombination sequence X,
- the unit U1 being contiguous with U3b, this in turn being contiguous with the units nxU2a, these in turn being contiguous with the units mxU2b, these in turn being contiguous with the unit U3a, this in turn being contiguous with the unit U3c.
A double-stranded DNA vector with no multiple cloning site and allowing elimination of the host cells having integrated one or more of the transgenes by non-homologous recombination and consisting of a sequence comprising the following functional units:
- U1, U3b, nxU2a, mxU2b, U3a, U3c and U3d,
- U1 representing a bacterial functional unit,
- U3b representing motif 5′ of a homologous recombination sequence X
- U2a representing an expression functional unit of which the promoter is dependent on RNA polymerase II and of which the expression product is a protein,
- U2b representing an expression functional unit of which the promoter is dependent on RNA polymerase III and of which the expression product is a non-coding RNA,
- n being greater than or equal to 0,
- m being greater than or equal to 0, on the condition that n+m≥2,
- U3a representing a positive selection cassette,
- U3c representing motif 3′ of a homologous recombination sequence X,
- U3d representing a negative selection cassette,
- the unit U1 being contiguous with the unit U3b, this in turn being contiguous with the units nxU2a, these in turn being contiguous with the units mxU2b, these in turn being contiguous with the unit U3a, this in turn being contiguous with the unit U3c, this in turn being contiguous with the unit U3d.
A double-stranded DNA vector with no multiple cloning site and allowing expression of one or more transgenes in an inducible manner and consisting of a sequence comprising the following functional units:
- U1, U2c, nxU2d, and mxU2e,
- U1 representing a bacterial functional unit,
- U2c representing a gene coding a transcriptional transactivator,
- U2d representing a gene of which the promoter is dependent on the transactivator coded by the gene U2c,
- U2e representing a gene of which the promoter is not dependent on the transactivator coded by the gene U2c,
- n being greater than or equal to 1,
- m being greater than or equal to 0,
- the unit U1 possibly being contiguous with the unit U2c, this in turn possibly being contiguous with the units nxU2d, these in turn possibly being contiguous with the units mxU2e,
- the unit U1 preferably being contiguous with the unit U2c, this in turn preferably being contiguous with the units nxU2d, these in turn preferably being contiguous with the units mxU2e.
A double-stranded DNA vector with no multiple cloning site and allowing execution of the genetic complementation under inducible control and consisting of a sequence comprising the following functional units:
- U1, U2f, U2c, and U2g,
- U1 representing a bacterial functional unit,
- U2c representing a gene coding a transcriptional transactivator,
- U2f representing a gene of which the promoter is an RNA polymerase III promoter and of which the expression product is a short hairpin RNA (shRNA) precursor of a small interfering RNA targeting a gene X,
- U2g representing a gene of which the promoter is dependent on the transactivator coded by the gene U2c and of which the expression product is a mutated version of the product of the gene X so as to be insensitive to the product of the gene U2f,
- the unit U1 possibly being contiguous with the unit U2f, this in turn possibly being contiguous with the unit U2c, this in turn possible being contiguous with the unit U2g,
- the unit U1 preferably being contiguous with the unit U2f, this in turn preferably being contiguous with the unit U2c, this in turn preferably being contiguous with the unit U2g.
A double-stranded DNA vector with no multiple cloning site and allowing selection of the cells of which the genome has been edited by targeted homologous recombination and consisting of a sequence comprising the following functional units:
- U1, U3a, U3b and U3c,
- U1 representing a bacterial functional unit,
- U3a representing a positive selection cassette,
- U3b representing motif 5′ of a sequence of homologous recombination X
- U3c representing motif 3′ of a homologous recombination sequence X,
- the unit U1 being contiguous with the unit U3b, these in turn being contiguous with the unit U3a, this in turn being contiguous with the unit U3c.
Dependencies are inferred from the claim text.