JP2002528129A - Recombinant method and reagents useful for it - Google Patents

Abstract

  (57) [Summary] The present invention generally relates to a method for recombination of at least two nucleic acid sequences and reagents useful for the method. More particularly, the present invention relates to a method for recombination of a circular nucleic acid sequence with a linear nucleic acid sequence in a host cell expressing recBCD nuclease or a functional derivative thereof. The method of the present invention is particularly useful for modifying a bacterial artificial chromosome with a linear DNA sequence by homologous recombination.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention generally relates to methods for recombination of at least two nucleic acid sequences and reagents useful for the methods. More particularly, the present invention relates to a method for recombination of a circular nucleic acid sequence with a linear nucleic acid sequence in a host cell expressing recBCD nuclease or a functional derivative thereof. The method of the present invention is particularly useful for modifying a bacterial artificial chromosome with a linear DNA sequence by homologous recombination.

BACKGROUND OF THE INVENTION [0002] The details of the publications referred to by number in this specification are collected at the end of the description. There is a need to better understand the structure and organization of higher eukaryotic genomes. Genomic sequences are often much longer than coding sequences. Although the function of most non-coding sequences is not yet known, it is clear that they play important roles in the regulation, stability and evolution of gene expression. In contrast to cDNA constructs driven by irrelevant or virus-derived promoters, gene transfer of large genomic fragments shows accurate temporal- and tissue-specific gene expression ^1-7 .

[0003] Yeast artificial chromosomes (YACs) have provided genomic clones of many disease genes ^8-10 . Transgenic models with YACs for many human loci have shown the utility of such large genomic fragments in generating accurate models of various diseases ^11-14 . High efficiency of homologous recombination in yeast, functional YAC genetic manipulation for analysis ^3,8,15 larger Y from YAC to cyclize ^16,17 and smaller overlapping YAC to PAC or BAC
It is very important in the generation ¹⁸ of AC.

[0004] However, the YAC system has several limitations, which have severely hindered its use. YAC has a high degree of clonal instability and chimerism ^19,20 . In addition, although several methods have been developed to generate YAC transgenics, it is difficult to purify complete YAC DNA for transgenesis ²¹ . An important deficiency of the yeast homologous recombination system in introducing modifications into the YAC or on TAR cloning is the difficulty in controlling the degree of homologous recombination, ²² and the correct product. A large number of "recombinant" clones need to be screened to identify

[0005] The large insertion BAC / PAC cloning systems ²³ , ²⁴ overcome many of the difficulties of the YAC system. BAC / PAC is used increasingly long range physical mapping ^{25, 26,} positional cloning ²⁷ disease genes, whole genome sequencing projects ^28-30 and functional studies ^31,32. High quality BAC / PAC genomic libraries are much easier to construct than YAC libraries due to their higher cloning efficiency in bacteria. BAC / PAC is
It is maintained at 1-2 copies per cell in a well-defined recombination-deficient E. coli strain DH10B and shows high clonal stability over many generations. BAC / PA
C carries inserts up to about 300 kb in size, but can be purified in large quantities for functional studies by conventional bacterial plasmid isolation methods. The genomic insert in these clones is large enough to preserve the integrity of most human loci,
It is therefore ideal for functional research.

[0006] BAC / PAC has the above advantages as an important tool for conducting genomic studies in many systems, and despite its increasing popularity, is a convenient tool for performing genetic manipulations on these clones. Technology does not exist. The large size of the genomic insert in these clones precludes the discovery of convenient restriction sites in routine operation. Dr. overlapping using homologous recombination of F plasmid based vector in E. coli
Ligation of the osophila cosmid fragment to generate a 125 kb fragment was performed in 1989.
First performed ³⁵ years. Co-integration between shuttle plasmids with a temperature-sensitive origin of replication in the rec ⁺ strain required a second site-specific recombination event in the rec ^- host to separate the co-integrant. Sternberg ³⁴ describes transposition insertion of a eukaryotic selectable marker into a large P1 clone, which may be difficult to control precisely at the translocation site, but should be applicable to PAC and BAC. is there. RecA-assisted restriction endonuclease (RARE) cleavage has also been used successfully to introduce modifications into many P1 and BAC clones, ³⁵ It is a very limited method. Since both the PAC and BAC cloning systems contain a loxP site on the backbone of each vector, many researchers have used cre recombinase to target the insertion of eukaryotic selectable markers and reporter genes into this site ^{. 37} .

[0008] Yang et al. ³⁸ report that wild-type r in a shuttle plasmid having a temperature-sensitive origin of replication as a means of imparting transient recombination proficiency to DH10B cells.
We report a targeted modification of a 131 kb BAC using the ecA gene. However, there was evidence that it was necessary to characterize the recombinant clones by Southern blotting to identify the correct product in order to perform significant rearrangement of the clones using this method. Another method using the CBTS strain of E. coli that contains a temperature-sensitive ambar mutation in the recA gene is also available from the complete mouse CMV
³⁹ has been used to introduce modifications into the 230 kb BAC, including the genome. However, direct transmission of BAC from DH10B to a conditional recA strain is difficult. Recently, using a targeting construct containing a chi site in the appropriate orientation, the green fluorescent protein gene has a zebrafish BA containing the GATA-2 locus.
⁴⁰ targeted in C. However, this method requires that the BAC be transferred to a strain capable of recombination, which can lead to instability in clones with repetitive sequences.

[0008] A method for substituting the RecBCD function of E. coli with the Red-Gam system of bacteriophage λ has also been described ⁴¹ . This substitution made it possible to carry out homologous recombination between the host chromosome and the short DNA fragment at a rate at least 70 times higher than that exhibited by the recBC sbsBC or recD strain. However, the usefulness of this system in modifying BAC / PAC has not been studied.

(Disclosure of the Invention) (Technical Problems to be Solved by the Invention) None of the above methods is optimal for genetic manipulation of PAC and BAC. These methods are not directly applicable to host cells such as DH10B expressing RecBCD , or require the construction of complex shuttle vectors. The degree of homologous recombination is also not well controlled in most of these methods, and therefore carries the risk of unwanted rearrangements. Accordingly, there is a need to develop a method for recombination of two nucleic acid sequences, such as PAC / BAC and linear DNA sequences, directly and in a controlled manner in a host cell.

In the course of the research leading up to the present invention, we sought to develop a method for the controlled in vivo homologous recombination of two nucleic acid sequences, such as a circular DNA sequence and a linear DNA sequence. did. In particular, we can modulate in host cells (modula
Table) BACs were modified by expressing the recE / recT recombination system with modulatable levels of the recBCD nuclease inhibitor to allow homologous recombination of the DNA sequence with BAC. Expression of the recBCD nuclease inhibitor acts to inhibit the functional activity of endogenously produced recBCD nuclease. Furthermore, the recE / recT homologous recombination system and re
The ability to modulate and match the expression of both with the cBCD nuclease inhibitor facilitates the control of homologous recombination events and minimizes random and unwanted recombination events. The ability to modulate and match the expression of both a homologous recombination system and a recBCD nuclease inhibitor can be demonstrated in vivo when at least one of the nucleic acid sequences subjected to recombination is located in an F plasmid, for example, BAC. Or where the host cell is sensitive to recombination-based molecules, or overexpression of a recBCD inhibitor such as gam, to facilitate induction of homologous recombination events.

(Solution method) [0011] The sequence identification number (SE) of the nucleic acid sequence and the amino acid sequence referred to in the present specification.
Q ID NO) is defined after the literature. Accordingly, the invention is a method of facilitating homologous recombination between at least two nucleotide sequences, under conditions sufficient to facilitate homologous recombination of the at least two nucleotide sequences. recBCD , and exonuclease,
Culturing a host cell capable of expressing a nucleotide sequence encoding a recombinant protein and a recBCD inhibitor or derivative thereof, including inducing recombination between the at least two nucleotide sequences in the host cell, wherein the exonuclease comprises , The expression of the recombinant protein and the recBCD inhibitor can be modulated.

[0012] Another aspect of the invention is a method of facilitating homologous recombination between at least two nucleotide sequences, wherein the method is sufficient to facilitate homologous recombination of the at least two nucleotide sequences. Culturing a host cell capable of expressing a nucleotide sequence encoding recBCD , recE , recT , and a recBCD inhibitor or a derivative thereof under suitable conditions, thereby inducing recombination between the at least two nucleotide sequences in the host cell. And wherein the expression of the nucleotide sequence encoding said recE , recT and recBCD inhibitor is modulatable.

[0013] Yet another aspect of the invention is a method of facilitating homologous recombination between at least two nucleotide sequences, wherein the method facilitates homologous recombination of the at least two nucleotide sequences. Under sufficient conditions, recBCD , recE , recT
And inducing recombination between said at least two nucleotide sequences in said host cell by culturing a host cell capable of expressing gam or a functional derivative thereof, wherein expression of said recE , recT and gam is carried out. A method is provided that is capable of being modulated.

[0014] Yet another aspect of the invention is a method for facilitating homologous recombination between at least two nucleotide sequences, wherein the method facilitates homologous recombination of the at least two nucleotide sequences. Under sufficient conditions, recBCD , recE , recT
And culturing a host cell capable of expressing gam or a derivative thereof,
Providing a method comprising inducing recombination between said at least two nucleotide sequences in said host cell, wherein the expression of said recE , recT and gam is modulatable .

[0015] Yet another aspect of the invention is a method of facilitating homologous recombination between at least two nucleotide sequences, wherein the method facilitates homologous recombination of the at least two nucleotide sequences. By culturing , under sufficient conditions, a host cell capable of expressing recBCD , and a nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or a derivative thereof, the host cell may be capable of expressing between the at least two nucleotide sequences. There is provided a method comprising inducing recombination, wherein the expression of said exonuclease, recombinant protein and recBCD inhibitor is modulatable.

Yet another aspect of the present invention is a method for facilitating homologous recombination between a cyclic nucleotide sequence and a linear nucleotide sequence, the method comprising the steps of: homologous recombination under conditions sufficient to facilitate, re
By culturing a host cell capable of expressing cBCD , and a nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or a derivative thereof, the host cell can be used to separate the cyclic nucleotide sequence from the linear nucleotide sequence. There is provided a method comprising inducing recombination, wherein the expression of said exonuclease, recombinant protein and recBCD inhibitor is modulatable.

[0017] Still another aspect of the present invention relates to a method for preparing a cyclic nucleotide sequence and a linear nucleotide sequence.
A method of facilitating homologous recombination between the cyclic nucleotide sequence and the
Under conditions sufficient to facilitate homologous recombination with the linear nucleotide sequence,re
cBCD,recE,recT,andgamOr express a functional derivative thereof
DH10B cells or homologs or mutants thereof are cultured,
DH10B cells or homologs or variants thereof, wherein said cyclic nucleotide sequence is
Inducing recombination with a linear nucleotide sequence.recBCD, recE ,recT,andgamIs characterized by its ability to be modulated
Provide a way to

[0018] Yet another aspect of the invention is a method of facilitating homologous recombination between at least two nucleotide sequences wherein at least one nucleotide sequence comprises an F plasmid portion, said at least two nucleotide sequences comprising: By culturing a host cell capable of expressing recBCD , and nucleotide sequences encoding exonuclease, recombinant protein and recBCD inhibitor or derivative thereof under conditions sufficient to facilitate homologous recombination of the sequence, Providing a method comprising inducing recombination between said at least two nucleotide sequences in said host cell, wherein expression of said exonuclease, recombinant protein and recBCD inhibitor is modulatable.

Yet another aspect of the invention is a method for facilitating homologous recombination between a BAC and a linear nucleotide sequence, the method comprising the step of homologous recombination between the BAC and the linear nucleotide sequence. the under conditions sufficient to facilitate, recBCD, and exonuclease, by culturing host cells capable of expressing the nucleotide sequence encoding the recombinant protein and recBCD inhibitor or a derivative thereof, the BAC in the host cell And inducing recombination between the exonuclease, the recombinant protein and the recBCD inhibitor, the method comprising directing recombination between the exonuclease and the linear nucleotide sequence.

Another aspect of the present invention is a method for facilitating homologous recombination of at least two nucleotide sequences in a host cell, comprising the steps of: (i) transforming the host cell, The transformed host cell comprises an exonuclease, a recombinant protein and a first nucleotide sequence encoding a recBCD inhibitor or a functional derivative thereof, and the at least two nucleotide sequences, wherein the exonuclease, the recombinant protein, and the recBC
Expression of the gene encoding the D inhibitor can be modulated, and (ii) culturing the transformed host cell for a time and under conditions sufficient for expression of the first nucleotide sequence to occur; In that case, the expression product of the first nucleotide sequence includes a method comprising: facilitating homologous recombination between the second nucleotide sequence and the third nucleotide sequence.

Yet another aspect of the invention is a method of producing a microorganism useful for facilitating homologous recombination between at least two nucleotide sequences, comprising recBCD , a modulatable level of an exonuclease, Methods that include genetically engineering a host cell to allow expression of a recombinant protein and a gene encoding a recBCD inhibitor and the at least two other nucleotide sequences.

[0022] Yet another aspect of the invention is a cell that can facilitate homologous recombination between at least two nucleotide sequences, wherein the nucleotide sequence encodes an exonuclease, a recombinant protein and a recBCD inhibitor, recBCD and Cells comprising the at least two nucleotide sequences and characterized in that the expression of the exonuclease, the recombinant protein and the recBCD inhibitor can be modulated.

Still another aspect of the present invention includes a nucleic acid molecule that has been homologously recombined by the method of the present invention. Another aspect of the invention is that one or more pharmacologically acceptable carriers and / or
Or a pharmaceutical composition comprising the modified nucleic acid molecule produced by the method of the present invention together with a diluent.

[0024] Yet another aspect of the invention is a kit for facilitating homologous recombination of at least two nucleotide sequences in a host cell, the kit comprising an exonuclease, a recombinant protein, a recBCD inhibitor or a functional thereof. A kit comprising one or more nucleotide sequences encoding a derivative, and a compartment adapted to contain reagents useful for facilitating homologous recombination. In addition, to accommodate biological samples such as, for example, one or more nucleotide sequences to be recombined, host cells or host cells already stably transformed with one or more nucleotide sequences to be recombined. Sections may be included.

[0025] Yet another aspect of the invention is a kit for facilitating homologous recombination of at least two nucleotide sequences in a host cell, the kit comprising an exonuclease, a recombinant protein, a recBCD inhibitor or a functional inhibitor thereof. A kit comprising a compartment adapted to accommodate derivatives and reagents useful to facilitate homologous recombination. In addition, to accommodate biological samples such as, for example, one or more nucleotide sequences to be recombined, host cells or host cells already stably transformed with one or more nucleotide sequences to be recombined. Sections may be included.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is directed, in part, to the introduction of nucleotide sequences expressing modulatable levels of exonucleases, recombinant proteins and recBCD nuclease inhibitors into host cells. Based on the development of a method for performing homologous recombination of at least two nucleotide sequences in a host cell. Expression of the recBCD nuclease inhibitor is required to inhibit the linear DNA degrading activity of recBCD nuclease expressed endogenously by host cells such as E. coli DH10B strain. Therefore, the method of the present invention is based on the modulatable and consistent induction of a homologous recombination system and the inhibition of recBCD nuclease activity. The emergence of a modulatable and consistent induction system facilitates, inter alia, the application of homologous recombination methods to host cells that are susceptible to overexpression of either one or more recombinant molecules or recBCD inhibitors. did. The method of the invention is also applicable, inter alia, when homologous recombination of a nucleic acid sequence comprising an F plasmid portion is desired.

Thus, the present invention is a method for facilitating homologous recombination between at least two nucleotide sequences, wherein the conditions are sufficient to facilitate homologous recombination between the at least two nucleotide sequences. Below, recBCD , and exonuclease,
Culturing a host cell capable of expressing a nucleotide sequence encoding a recombinant protein and a recBCD inhibitor or derivative thereof, including inducing recombination between the at least two nucleotide sequences in the host cell, wherein the exonuclease comprises , The expression of the recombinant protein and the recBCD inhibitor can be modulated.

Reference to “exonuclease” refers to exposing a single-stranded region on a double-stranded nucleic acid sequence (such a single-stranded region forms a pair between this region and the complementary region of another nucleic acid sequence). (Required to cause recombination) to facilitate homologous recombination. Exonucleases are those that are suitable for use with the selected recombinant protein. Examples of exonucleases suitable for use in the present invention include, but are not limited to, a Redλ recombination exonuclease called exo, and recE / r called recE.
ecT recombinant exonuclease, or a functional derivative thereof. Preferably, the exonuclease is recE or a functional derivative thereof. Reference to "recombinant protein" refers to mediating recombination between a single-stranded region of one nucleic acid sequence (such as a single-stranded region generated by an exonuclease) and a complementary region of another nucleic acid sequence. Should be understood as a reference to a peptide, polypeptide or protein that facilitates homologous recombination. Examples of recombinant proteins include, but are not limited to, Redλ recombinant protein bet and recE / recT recombinant protein recT, or functional derivatives thereof. The nucleotide sequences encoding recE and recT are referred to herein as recE and recT , respectively.

Thus, the present invention more particularly relates to a method for the synthesis of at least two nucleotide sequences.
For facilitating homologous recombination of the at least two nucleotide sequences.
Under conditions sufficient to facilitate homologous recombination of the sequence,recBCD,recE, recT And a recBCD inhibitor or a functional derivative thereof
Culturing a host cell capable of expressing the nucleotide sequence
Inducing recombination between said at least two nucleotide sequences.re
cE,recTAnd nucleotide sequences encoding recBCD inhibitors
Is modulatable.

The term “expression” refers to the transcription and translation of a nucleotide sequence that results in the synthesis of a peptide, polypeptide or protein. Reference to "a gene encoding a recBCD inhibitor" refers to inhibiting a recBCD nuclease or sufficiently inhibiting its activity such that a linear DNA sequence is not degraded by the recBCD nuclease, thereby allowing homologous recombination to occur. It should be understood as a reference to a nucleotide sequence that encodes an expression product that can be down-regulated. R suitable for use in the present invention
Examples of ecBCD inhibitors include, but are not limited to, ga
m, P22Abc protein or SSB protein or a functional derivative thereof. Preferably, the recBCD inhibitor is gam. ga
The nucleotide sequence encoding m is referred to herein as gam .

According to a preferred embodiment of the present invention, the present invention relates to a method for facilitating homologous recombination between at least two nucleotide sequences, said method facilitating homologous recombination between said at least two nucleotide sequences. Under conditions sufficient to make recBCD , re
culturing a host cell capable of expressing cE , recT , and gam or a functional derivative thereof, including inducing recombination between said at least two nucleotide sequences in said host cell, wherein said recE , recT, and gam Provides a method characterized in that the expression of is modulatable

[0032] Reference to "host cell" herein should be understood as a reference to any prokaryotic or eukaryotic cell that can be transformed or transfected with the nucleic acid sequence. For example, encompassed by the present invention is the cloning of nucleotide sequences, such as host cells used to generate gDNA or cDNA libraries or host cells used to clone vectors containing the desired DNA sequence insert. And / or a host cell suitable for expression. According to the present invention, the host cell preferably expresses recBCD nuclease (also referred to as "exonuclease V"; particularly, it degrades linear double-stranded DNA). A host cell may express a recBCD by expression of a naturally occurring recBCD gene or by transforming or transfecting the host cell with a nucleic acid molecule encoding the recBCD .

Without limiting the present invention to any one theory or mechanism of action, the present inventors have set forth certain strains of host cells in which one or more of a recBCD inhibitor and / or a homologous recombination system is used. Is sensitive to overproduction, for example, constitutive production, of the molecule. “Sensitive” means that the host cell is impaired in some way, for example, reduced in its viability or one or more undesired modulations of its functional activity. For example, rec in DH10B host cells
Constitutive production of the BCD inhibitor, gam, has been found to be toxic in that it reduces the viability of DH10B cells. The development of recombination molecules and in vivo recombination systems capable of modulating and matching the expression of recBCD inhibitors has overcome this toxicity problem. Thus, in a preferred embodiment, the host cell is rec.
A host cell that is sensitive to one or more constitutive productions of a BCD inhibitor, exonuclease or recombinant protein. Even more preferably,
The host cell is DH10B or a variant or homolog thereof. The invention also encompasses the use of host cell variants and homologs.

According to this most preferred aspect, the present invention is a method for facilitating homologous recombination between at least two nucleotide sequences, said method facilitating homologous recombination of said at least two nucleotide sequences. Culturing a host cell capable of expressing recBCD , and a nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or derivative thereof, under conditions sufficient to produce the at least two nucleotides in the host cell. Inducing recombination between sequences, said exonuclease, recombinant protein and recBCD
A method is provided wherein the expression of the inhibitor is modulatable. Even more preferably, the exonuclease is recE, the recombinant protein is recT, and the recBCD inhibitor is gam.

[0035] Reference to "at least two nucleotide sequences" should be understood as a reference to two or more nucleotide sequences to be homologously recombined by the method of the present invention. These nucleotide sequences may be separate nucleic acid molecules, such as, for example, two circular vectors (eg, two plasmids, etc.), a plasmid and a linear DNA sequence, or a chromosome and a linear DNA sequence. In another embodiment, the nucleotide sequences are two portions of a single nucleic acid molecule, such as a linear nucleotide sequence portion and a cyclic nucleotide sequence portion of a single nucleotide molecule, such as occurs during rolling circle replication. May be. Preferably,
These nucleotide sequences are a cyclic nucleotide sequence and a linear nucleotide sequence. In this context, "cyclic" nucleotide sequence should be understood as a reference to the cyclic nucleotide sequence portion of any nucleotide molecule. For example, the cyclic nucleotide sequence may be completely circular, such as a plasmid, or partially circular, such as the cyclic portion of a nucleotide molecule that occurs during rolling circle replication. In this context, a "cyclic" nucleotide sequence corresponds to the cyclic portion of the molecule. Preferably, the circular nucleic acid sequence is an artificial chromosome of the BAC or PAC type.

A “linear” nucleotide sequence should be understood as a reference to any nucleotide sequence that is in an essentially linear form. The linear sequence may be a linear nucleotide molecule, or may be a linear portion of a nucleotide molecule that also includes non-linear portions such as cyclic portions. Examples of linear nucleotide sequences include, but are not limited to, PCR products, cleavage products, synthetic DNA, or linear portions of nucleotide molecules that occur during rolling circle replication. Recombination of a linear nucleotide sequence with a cyclic nucleotide sequence may, for example, introduce a selectable marker or a particular mutation in the cyclic nucleotide sequence. The homologous recombination of the invention may occur between nucleotide sequences introduced into the cell, or may occur between a nucleotide sequence naturally found in the cell and one or more introduced nucleotide sequences. .

Accordingly, the present invention preferably provides a method for facilitating homologous recombination between a cyclic nucleotide sequence and a linear nucleotide sequence, the method comprising the steps of: the recombination under conditions sufficient to facilitate, r
ecBCD , and exonucleases, recombinant proteins and recBCD
Culturing a host cell capable of expressing a nucleotide sequence encoding an inhibitor or a derivative thereof to induce recombination between said cyclic nucleotide sequence and said linear nucleotide sequence in said host cell, A method is provided wherein the expression of the nuclease, the recombinant protein and the recBCD inhibitor is modulatable. Preferably, the host cell is a DH10B cell. Even more preferably, the exonuclease is recE, the recombinant protein is recT, and the recBCD inhibitor is gam or a functional derivative thereof.

According to a preferred embodiment, the present invention relates to a method for facilitating homologous recombination between a cyclic nucleotide sequence and a linear nucleotide sequence, comprising the steps of: Culturing DH10B cells or homologs or variants thereof capable of expressing recBCD , recE , recT , and gam or a functional derivative thereof under conditions sufficient to facilitate homologous recombination of the DH10B. Inducing recombination between said cyclic nucleotide sequence and said linear nucleotide sequence in a cell or homolog or mutant thereof, wherein said recB
A method is provided wherein the expression of CD , recE , recT , and gam is modulatable .

Although the nucleic acid sequences of the present invention are preferably derived from the human genome, genomic and nucleotide sequences from non-human animals and plants are also encompassed by the present invention. Non-human animals encompassed by the present invention include primates, livestock animals (eg, sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (eg, mice, rats, guinea pigs, hamsters, rabbits) ), Domesticated pet animals (eg, dogs, cats), birds (eg, chickens, geese, ducks and other poultry, gamebirds, emu, ostriches) and captive wild or captive Animals (eg, fox, kangaroo, dingo).

The inventors have determined that the homologous recombination system of the invention is suitable for recombining at least two nucleotide sequences such that at least one of the at least two nucleotide sequences comprises an F plasmid portion. It was determined. A reference to a nucleotide sequence that “contains” an F plasmid portion refers to a fusion to all or part of the F plasmid,
It should be understood as a reference to a nucleotide sequence that is linked or otherwise associated. For example, the nucleotide sequence of interest may be located in a vector, the backbone of which contains sequences corresponding to all or part of the F plasmid. An F plasmid-based BAC contains, for example, a wild-type F plasmid origin of replication and a genomic sequence of interest located in a vector that expresses the parA, parB and parC coding nucleotide sequences.

Thus, in another preferred embodiment, the present invention relates to a method for facilitating homologous recombination between at least two nucleotide sequences wherein at least one nucleotide sequence comprises an F plasmid portion, said method comprising: Culturing a host cell capable of expressing recBCD , and a nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or derivative thereof, under conditions sufficient to facilitate homologous recombination of the two nucleotide sequences. Thereby inducing recombination between said at least two nucleotide sequences in said host cell, wherein the expression of said exonuclease, recombinant protein and recBCD inhibitor is modulatable. provide. Preferably, the nucleotide sequence containing the F plasmid portion is BAC. Even more preferably, the host cell is DH10B or a variant or homologue thereof.

The BAC / PAC cloning system has facilitated the study of mammalian genomes by gene transfer of large genomic fragments exhibiting accurate time and tissue-specific gene expression. Therefore, large insertion BAC / PAC cloning systems are widely used for long-range physical mapping, positional cloning of disease genes, whole-genome sequencing projects and functional studies. Therefore, BAC /
The PAC E. coli library was used for human genomic DNA as well as baboons, dogs,
It has been produced for genomic DNA of other animal and plant species, including cattle, sheep, goats and rice. BAC / PAC is generally maintained at 1-2 copies per cell in a well-defined recombination deficient E. coli strain DH10B. Due to the interest in modifying BACs and PACs by introducing linear DNA segments, a particularly preferred embodiment of the present invention is to modify BACs or PACs by homologous recombination of the linear DNA segments into BACs or PACs. About.

According to this most preferred aspect, the present invention is a method for facilitating homologous recombination between a BAC and a linear nucleotide sequence, the method comprising the steps of: Culturing a host cell capable of expressing recBCD and nucleotide sequences encoding exonucleases, recombinant proteins and recBCD inhibitors or derivatives thereof under conditions sufficient to facilitate targeted recombination. Inducing recombination between the BAC and the linear nucleotide sequence with the exonuclease, the recombinant protein and recBC.
A method is provided wherein the expression of the D inhibitor is modulatable. Preferably, the host cell is a DH10B cell or a mutant or homolog thereof. Even more preferably, the exonuclease is recE, the recombinant protein is recT, and the recBCD inhibitor is gam.

In yet another most preferred embodiment, the present invention is a method for facilitating homologous recombination between a PAC and a linear nucleotide sequence, said method comprising: RecBCD under conditions sufficient to facilitate targeted recombination
And culturing a host cell capable of expressing a nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or a derivative thereof, to allow recombination between the PAC and the linear nucleotide sequence in the host cell. Inducing the exonuclease, recombinant protein and re
A method is provided wherein the expression of the cBCD inhibitor is modulatable. Preferably, the host cell is a DH10B cell or a mutant or homolog thereof. Even more preferably, the exonuclease is recE, the recombinant protein is recT, and the recBCD inhibitor is gam.

The method of the present invention may comprise one or morerecBCD, Exonuclease and
Sequence encoding recombinant and recombinant proteins, recBCD inhibitor
Or a nucleotide sequence encoding a derivative thereof and said at least two nucleotides
And transforming a host cell with the leotide sequence. Easy operation of the method of the invention
How many of these molecules need to be introduced into the host cell to
Will depend on the particular case in question. For example, the illustrated rec
For the E / recT recombination system, commercially available BAC or PAC DH10B
It is desirable to use a population of host cells from the library. BAC or PAC molecule
After transformation with nucleic acid molecules encoding recE, recT and gam
Linear nucleoti introduced into a commercially available DH10B clone from the library
It can be modified by recombination with the nucleotide sequence. DH10B cells are endogenous
TorecBCDIs expressed. Alternatively, alreadyrecBCD,recE, recT And nucleotide sequences encoding recBCD inhibitors
If a host cell that has undergone homologous recombination is still present in the host cell,
It is only necessary to transform with the missing nucleotide sequence. These host cells
,recBCD,recE,recTAnd recBCD inhibitor code
The nucleotide sequence to be expressed to express these one or more molecules.
Either genetically engineered cells or cells that endogenously express these molecules
Can be expressed by

According to the method of the present invention, exonucleases, recombinant proteins and rec
BCD inhibitor expression can be modulated. This allows for transient and consistent expression of these proteins. "May be modulated" means that the expression of genes encoding these proteins can be up-regulated or down-regulated at the transcriptional or translational level. This modulation can be achieved by any of a number of techniques including, but not limited to, controlling promoter expression or controlling methylation. For example, as exemplified herein, DH10B cells transformed with the BAC nucleic acid molecule pEBAC140 are transformed into an inducible expression plasmid pGETrec (inducible L-
(Including nucleic acid molecules encoding recE, recT and gam under the control of the arabinose promoter). By culturing these cells in the presence of L-arabinose, the expression of the pGETrec plasmid is induced. Then, when these cells were electroporated with a double-stranded linear DNA such as PCRkan140, the recE / recT homologous recombination system and the simultaneous inhibition of the recBCD nuclease activity by the gam expression product resulted in P.
Homologous recombination of CRkan140 and pEBAC140 occurs. Once homologous recombination is complete, expression of the pGETrec plasmid can be down-regulated by removing DH10B cells from the L-arabinose-rich medium. This serves to stop the activity of the recE / recT homologous recombination pathway.

Illustrative of the invention are nucleic acid sequencesrecE,recTandgamSingle plus including
The mid under the control of an inducible promoter (ie, recE, recT and g
am encoding nucleic acid molecules are operably linked to a common promoter)
The method of the present invention was carried out by introducing into a host cell.recE,recT
andgamShould also include the use of two or more vectors which separately contain
Must understand. However,recE,recTandgamSimultaneous
To achieve consistency with respect to expression, for example,recE,recTand gam By using the same inducible promoter for each ofrecE,r
ecTandgamIt is necessary to ensure that the induction of
You. Therefore, the recE / recT homologous recombination system is a recBCD nuclease.
It could function simultaneously with gam inhibition of activity.

[0048] "Can facilitate" homologous recombination should be understood to mean inducing, promoting or otherwise contributing to the functional operation of a homologous recombination system. Must. For example, the recE and recT expression products are recE / re
Induces cT-based recombination, while the gam expression product inhibits the activity of recBCD nuclease, which disrupts recombination by degrading linear DNA.

Without limiting the invention to any one theory or mechanism of action, pGETr
ec can stably coexist with BAC / PAC in E. coli DH10B cells. pGET
By using rec, homologous recombination between a linear nucleic acid fragment and a target circular nucleic acid molecule can be performed with high efficiency. The efficiency of homologous recombination increases as the length of homology increases from 50 bp to 140 bp. Further, it is believed that a significant increase in the efficiency of homologous recombination with BAC / PAC can be achieved by using non-contiguous sequences in the targeted homology region.

The present invention provides for introducing homologous recombination into a host cell, rather than introducing a nucleic acid molecule that is induced to express the protein or a functional derivative thereof in the host cell. It should be understood that this also encompasses the induction of homologous recombination in vivo by introducing a facilitating protein or a functional derivative thereof. For example, a consistent introduction of the recE, recT and gam proteins or functional derivatives thereof and the nucleotide sequence to be recombined into the host cell is conceivable. It is also conceivable to carry out the method of the present invention by expressing some of the proteins of the homologous recombination system in a host cell and introducing the remaining proteins into the host cell in the form of proteins. For example, expression of recE and recT or a functional derivative thereof in a host cell may be performed with the consistent administration of the gam protein. It should be understood that this aspect of the invention also encompasses the delivery of any one or more of an exonuclease, a recombinant protein or a recBCD inhibitor or a functional derivative thereof.

“Derivatives” include fragments, parts, moieties, chemical equivalents, variants, homologs, analogs, mimetics from natural, synthetic or recombinant sources, including fusion proteins. Derivatives may be by amino acid insertion, deletion or substitution. Insertional derivatives of amino acids include fusions at the amino and / or carboxy terminus as well as intrasequence insertions of single or multiple amino acids. Insertion amino acid sequence variants are those in which one or more amino acids have been introduced at a predetermined site in the protein, but random insertion is also possible if the resulting product is screened appropriately. is there. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substituted amino acid variants have at least one residue in the sequence removed,
A different residue has been inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.

Derivatives of the nucleotide sequence and the expression product (referred to as “component”) include:
Fragments with particular epitopes or portions of all components fused to a peptide, polypeptide or other proteinaceous or non-proteinaceous molecule are included.
For example, the component or derivative thereof may be fused with a molecule that facilitates entry into a cell. Analogs of such components contemplated in the present invention include, but are not limited to, modification of side chains, introduction of unnatural amino acids and / or derivatives thereof during synthesis of peptides, polypeptides or proteins, and protein And the use of cross-linking and other methods that impart a conformational constraint to the sex molecule or analog thereof. Derivatives of nucleic acid sequences may also be by single or multiple nucleotide substitutions, deletions and / or additions, including fusions with other nucleic acid molecules. Derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co-suppression and fusion of nucleic acid molecules.

Examples of side chain modifications contemplated by the present invention include: reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ₄ ; amidination with methylacetimidate; acylation with acetic anhydride. Carbamoylation of amino groups with cyanate esters; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxal −5-
Modification of the amino group due to reduction with NaBH ₄ after pyridoxal the lysine phosphate.

The guanidine group of an arginine residue can be modified by using a reagent such as 2,3-butanedione, phenylglyoxal and glyoxal to form a heterocyclic condensation product. The carboxyl group can be modified via carbodiimide activation via formation of an O-acylisourea and subsequent derivatization, for example, to the corresponding amide.

Sulfhydryl groups can be carboxymethylated with iodoacetic acid or iodoacetamide; formic acid oxidation to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides;
Production of mercury derivatives using 4-chloromercuribenzoic acid ester, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercury compounds; with cyanate ester at alkaline pH It can be modified by a method such as carbamoylation.

Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halide. Tyrosine residues, on the other hand, can be altered by nitrating with tetranitromethane to form a 3-nitrotyrosine derivative. Modification of the imidazole ring of the histidine residue can be performed by alkylation using an iodoacetic acid derivative or N-carboethoxylation using diethyl pyrocarbonate.

Examples of unnatural amino acids and derivatives introduced during protein synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-
3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or Or the D-isomer of an amino acid. Table 1 shows a list of unnatural amino acids considered in the present invention.

Table 1 Uncommon amino acids Code α-aminobutyric acid Abu α-amino-α-methylbutyric acid Mgabu aminocyclopropanecarboxylate Cpro aminoisobutyric acid Aib aminonorbornylcarboxylate Norb cyclohexylalanine Chexa cyclopentylalanine Cpen D-alanine Dal D-arginine Darg D-aspartic acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamic acid Dglu D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-lysine D-lysine D-lysine D-lysine -Proline Dpro D-Serine Dser D-Threonine Dthr D-Tryptophan D rp D- tyrosine Dtyr D- valine Dval

D-α-methylalanine Dmala D-α-methylarginine Dmarg D-α-methylasparagine Dmasn D-α-methylaspartic acid Dmap D-α-methylcysteine Dmcys D-α-methylglutamine Dmgln D-α- Methylhistidine Dmhis D-α-methylisoleucine Dmile D-α-methylleucine Dmleu D-α-methyllysine Dmlys D-α-methylmethionine Dmmet D-α-methylornithine Dmorn D-α-methylphenylalanine Dmproline Dmpro D-α-methylserine Dmser D-α-methylthreonine Dmthr D-α-methyltryptophan Dmtrp D-α-methyltyrosine Dmty D-α-methylvaline Dmval

DN-methylalanine DNmala DN-methylarginine DNarg DN-methylasparagine DNmasn DN-methyl-aspartic acid DNmapsp DN-methylcysteine Dnmcys DN-methylglutamine Dnmgln D-N- Methylglutamic acid Dnmgl DN-methylhistidine Dnmhis DN-methylisoleucine Dnmile DN-methylleucine Dnmleu DN-methyllysine Dnmlys N-methylcyclohexylalanine Nmchexa D-N-methylornithine N-methylglycine Methylaminoisobutyric acid Nmaib N- (1-methylpropyl) glycine Nile N- (2-methylpropyl) glycine Nleu DN-methyl Tryptophan Dnmtrp D-N- methyl-tyrosine Dnmtyr D-N- methylvaline Dnmval

Gamma-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine Etg L-homophenylalanine Hphe L-α-methylarginine Marg L-α-methylaspartic acid Masp L-α-methylcysteine Mcys L-α -Methylglutamine Mgln L-α-Methylhistidine Mhis L-α-Methylisoleucine Mile L-α-Methylleucine Mleu L-α-Methylmethionine Mmet L-α-Methylnorvaline Mnva L-α-Methylphenylalanine Mphe L-α -Methylserine Mser L-α-methyltryptophan Mtrp L-α-methylvaline Mval

N- (N- (2,2-diphenylethyl) Nnbhm carbamylmethyl) glycine 1-carboxy-1- (2,2-diphenyl Nmbc ethylamino) cyclopropane LN-methylalanine Nmala LN -Methylarginine Nmarg LN-methylasparagine Nmasn LN-methylaspartic acid Nmapp LN-methylcysteine Nmcys L-N-methylglutamine Nmgln L-N-methylglutamic acid Nmglu L-N-methylhistidine Nmhis -Methylisoleucine Nmile L-N-methylleucine Nmleu LN-methyllysine Nmlys LN-methylmethionine Nmmet LN-methylnorleucine Nmnle L-methylnorvaline Nmnva L-methylorni Tin Nmorn L-N-methylphenylalanine Nmphe L-N-methylproline Nmpro L-N-methylserine Nmser L-N-methylthreonine Nmthr L-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr L-N-methyl-valine -N-methylethylglycine Nmetg L-N-methyl-t-butylglycine Nmtbug

L-norleucine Nle L-norvaline Nva α-methyl-aminoisobutyric acid Maib α-methyl-γ-aminobutyric acid Mgabu α-methylcyclohexylalanine Mchexa α-methylcyclopentylalanine Mcpen α-methyl-α-naphthylalanine Manap α- Methylpenicylamine Mpen N- (4-aminobutyl) glycine Nglu N- (2-aminoethyl) glycine Naeg N- (3-aminopropyl) glycine Norn N-amino-α-methylbutyric acid Nmaabu α-naphthylalanine Anap

N-benzylglycine Nphe N- (2-carbamylethyl) glycine Ngln N- (carbamylmethyl) glycine Nasn N- (2-carboxyethyl) glycine Nglu N- (carboxymethyl) glycine Nasp N-cyclobutyl Glycine Ncbut N-cycloheptylglycine Nhepp N-cyclohexylglycine Nchex N-cyclodecylglycine Ncdec N-cyclododecylglycine Ncpod N-cyclooctylglycine Ncoct N-cyclopropylglycine NcproN-glycine N-cycloundecyl -Diphenylethyl) glycine Nbhm N- (3,3-diphenylpropyl) glycine Nbhe N- (3-guanidinopropyl) glycine Narg N- (1-hydroxyethyl) g Shin NTHR N-(hydroxyethyl) glycine Nser N- (imidazolylethyl) glycine Nhis N-(3- indolyl) glycine Nhtrp

N-methyl-γ-aminobutyric acid Nmgabu DN-methylmethionine Dnmmet N-methylcyclopentylalanine Nmcpen DN-methylphenylalanine Dnmphe DN-methylproline Dnmpro DN-methylserine Dnmser DN-methyl Threonine Dnmthr N- (1-methylethyl) glycine Nval N-methyl-α-naphthylalanine Nmanap N-methylpenicylamine Nmpen N- (p-hydroxyphenyl) glycine Nhtyr N- (thiomethyl) glycine Ncys

Penicylamine Pen L-α-methylalanine Mala L-α-methylasparagine Masn L-α-methyl-t-butylglycine Mtbug L-methylethylglycine Metg L-α-methylglutamic acid Mglu L-α-methylhomophenylalanine Mhphe N- (2-methylthioethyl) glycine Nmet L-α-methyllysine Mlys L-α-methylnorleucine Mnle L-α-methylornithine Morn L-α-methylproline Mpro L-α-methylthreonine Mthr L-α- Methyltyrosine Mtyr LN-methylhomophenylalanine Nmhphe N- (N- (33-diphenylpropyl) Nnhecarbamylmethyl) glycine

Crosslinkers can be used to establish a three-dimensional conformation, for example, bifunctional imide esters having a (CH ₂ ) _n spacer group (n = 1 to n = 6), glutaraldehyde, Homo-2 such as N-hydroxysuccinimide ester
A functional crosslinking agent, and usually an amino-reactive residue such as N-hydroxysuccinimide and a residue specifically reactive with other groups such as maleimide, dithio residue (SH) or carbodiimide (COOH). Heterobifunctional reagents containing Furthermore, for example, C _alpha and N _alpha - during the introduction of the methyl amino acids, introduction of double bonds between C _alpha atom and C _beta atoms of the amino acid, and the N-terminus and C-terminus,
The conformation of the peptide can be restricted by the introduction of a covalent bond, such as the formation of an amide bond between the two side chains or between the side chain and the N- or C-terminus.

(Efficient Effects Over the Prior Art) The method of the present invention makes it possible to introduce a desired modification at any position in a circular DNA clone such as BAC or PAC without causing unwanted rearrangement. Useful as a simple and efficient technique. Modifications, such as the introduction of selectable markers or specific mutations corresponding to those known to cause human disease, are examples where the methods of the invention can be applied. The method of the present invention uses the cloned BAC /
It reduces the need to transfer circular DNA molecules, such as PACs, to other E. coli for homologous recombination. The method of the present invention also reduces the need for making special shuttle vectors. This method is so simple that it can be used repeatedly on a single clone to form multiple changes. The methods of the invention can be modified to include a reporter gene or other desired sequence in a circular nucleic acid molecule.

To generate accurate cell and animal models for a particular mutation, it is necessary to minimize interference with normal genetic structure. The introduction of a particular mutation change into a gene structure can be introduced in two steps using the recombinant method of the present invention. First, a PCR product with a tetracycline or a second counterselectable marker (eg, ccd
B , sacB ), a cassette containing an antibiotic selection marker linked to it is introduced into the target site, which is then exactly knocked out in a second step using a genomic fragment having only the desired modifications. The correct product can easily be obtained by counterselecting clones that have lost the tetracycline gene or a second counterselectable marker ^33,39,39,43 . Depending on the target site for integration in circular DNA molecules, these methods monitor the effects of various sequence elements and mutations on the organization and developmental specificity of gene transcription, splicing and expression from a fully functional locus. A very sensitive assay system for These technologies facilitate the development of pharmacological approaches and the development of human artificial chromosomes aimed at changing gene expression with increased tissue and locus specificity as a means to alleviate or treat various human genetic diseases. To Generation of accurate animal models with normal and mutated functional loci also facilitates gene therapy, including gene replacement or correction of mutated loci.

Another aspect of the invention is a method for facilitating homologous recombination of at least two nucleotide sequences in a host cell, comprising the steps of: (i) transforming the host cell, The transformed host cell comprises an exonuclease, a recombinant protein and a first nucleotide sequence encoding a recBCD inhibitor or a functional derivative thereof, and the at least two nucleotide sequences, wherein the exonuclease, the recombinant protein, and the recBC
Expression of the gene encoding the D inhibitor can be modulated, and (ii) culturing the transformed host cell for a time and under conditions sufficient for expression of the first nucleotide sequence to occur; In that case, the expression product of the first nucleotide sequence includes a method comprising: facilitating homologous recombination between the second nucleotide sequence and the third nucleotide sequence.

Preferably, the exonuclease is recE, the recombinant protein is recT, and the gene encoding a recBCD inhibitor is gam . Even more preferably, said at least two nucleotide sequences are a nucleotide sequence comprising an F plasmid portion and a linear nucleotide sequence. Even more preferably, the nucleotide sequence comprising the F plasmid portion is BAC. More preferably, the host cell is DH10B or a variant or homolog thereof.

[0072] Yet another aspect of the invention is a method of producing a microorganism useful for facilitating homologous recombination between at least two nucleotide sequences, comprising recBCD , a modulatable level of an exonuclease, Methods that include genetically engineering a host cell to allow expression of a recombinant protein and a gene encoding a recBCD inhibitor and the at least two other nucleotide sequences. Preferably, the exonuclease is recE, the recombinant protein is recT, and the gene encoding a recBCD inhibitor is gam . Even more preferably, said at least two nucleotide sequences are a nucleotide sequence comprising an F plasmid portion and a linear nucleotide sequence. Even more preferably, the nucleotide sequence comprising the F plasmid portion is BAC. More preferably, the host cell is DH10B or a variant or homolog thereof.

Still another aspect of the invention is a cell that facilitates homologous recombination between at least two nucleotide sequences, wherein the nucleotide sequence encodes an exonuclease, a recombinant protein and a recBCD inhibitor, recBCD and Cells comprising the at least two nucleotide sequences and characterized in that the expression of the exonuclease, the recombinant protein and the recBCD inhibitor can be modulated. Preferably, the exonuclease is recE, the recombinant protein is recT, and the gene encoding a recBCD inhibitor is gam . Even more preferably, said at least two nucleotide sequences are a nucleotide sequence comprising an F plasmid portion and a linear nucleotide sequence. Even more preferably, the nucleotide sequence comprising the F plasmid portion is BAC. More preferably, the host cell is DH10B or a variant or homolog thereof.

Yet another aspect of the present invention includes a nucleic acid molecule that has undergone homologous recombination according to the method of the present invention. Preferably, the modified nucleic acid molecule is a modified BAC or a modified PAC. The invention also includes the use of the modified nucleic acid molecules in treating and / or diagnosing a patient. Treatment methods include gene therapy. The present invention also includes a screening method using the modified nucleic acid molecule. Accordingly, another aspect of the present invention includes a pharmaceutical composition comprising a modified nucleic acid molecule produced by a method of the present invention together with one or more pharmaceutically acceptable carriers and / or diluents. In a most preferred embodiment, there is provided an expression vector comprising a gene construct substantially as shown in Figure 3 or a functional derivative thereof.

[0075] Yet another aspect of the present invention is a kit for facilitating homologous recombination of at least two nucleotide sequences in a host cell, comprising a exonuclease, a recombinant protein, a recBCD inhibitor or a functional inhibitor thereof. A kit comprising one or more nucleotide sequences encoding a derivative, and a compartment adapted to contain reagents useful for facilitating homologous recombination. In addition, to accommodate biological samples such as, for example, one or more nucleotide sequences to be recombined, host cells or host cells already stably transformed with one or more nucleotide sequences to be recombined. Sections may be included.

[0076] Yet another aspect of the invention is a kit for facilitating homologous recombination of at least two nucleotide sequences in a host cell, comprising a exonuclease, a recombinant protein, a recBCD inhibitor or a functional thereof. A kit comprising a compartment adapted to accommodate derivatives and reagents useful for facilitating homologous recombination. In addition, to accommodate biological samples such as, for example, one or more nucleotide sequences to be recombined, host cells or host cells already stably transformed with one or more nucleotide sequences to be recombined. Sections may be included.

A kit according to this aspect of the invention should also be understood to include a kit comprising a combination of nucleotide sequences encoding one or more recombinant proteins or a combination of the recombinant proteins themselves. Further features of the present invention are described in more detail in the following non-limiting examples and / or figures. However, it should be understood that such detailed description is included herein for the purpose of illustrating the invention only.

Example 1 Inducibility of Homologous Recombination of a PCR Fragment to a Cyclic Substrate in E. coli DH10B
ecE pathway The recE pathway of homologous recombination between linear and circular substrates is based on the sbcA re
^42-45 active in cBC strains JC8679 and JC9604. This route does not depend on the recA ^46. The recT gene appears to play a role for recA in homologous sequence pairing ⁴⁷ . Chloramphenicol resistance (Cm ^r ) in these cells Kanamycin resistance (Km ^r ) with a 50 bp flanking homology arm into the backbone of the pEBAC140 vector
Accurate homologous recombination of the PCR fragment of the gene (PCRkan50) is obtained and the pE
BAC140 :: kan50 was generated. However, 20 from DH10B
The 0 kb pEBAC / 148-globin clone was converted to JC9 for subsequent modification.
604 could not be transferred to electrically competent cells. This was likely due to host restriction differences between these two strains. Since BAC / PAC is usually retained in DH10B cells,
DH having desired BAC / PAC rather than transferring C / PAC to other E. coli strains
It is desirable to carry out the modification directly in the 10B cells. However, DH10B (p
Electroporation of PCRkan50 into EBAC140) was performed using Cm ^r K
No ^mr recombinant was produced. In addition, PCRkan14 in DH10B cells
0 (1450 bp PC with about 140 bp flanking homology arm)
R fragment) to the pEBAC140 backbone could not be obtained.

The recE gene and recT under the control of an inducible arabinose promoter
The pBAD24-recET plasmid having the gene was transferred to DH10B (pEBAC
140) Electroporated into cells and clones containing both plasmids were selected on medium containing ampicillin and chloramphenicol. A single colony with both plasmids, DH10B (pBAD24-recET)
, PEBAC140) to induce L-arabinose during the growth of the cells to prepare electrically competent cells. Also in this case, PCRkan5 was added to these cells.
No Cm ^r Km ^r recombinant clones were obtained after electroporation of 0.

The plasmid pGETrec was prepared by inserting the PCR fragment containing the gam gene into the pBAD24-recET plasmid (FIG. 3). The gam gene is replaced with recE
Placing under the same L-arabinose promoter as the gene and the recT gene sought to obtain consistent induction of the recE pathway and inhibition of recBCD nuclease activity on subsequently introduced linear double-stranded DNA. This inducible expression plasmid pGETrec was introduced into electrocompetent DH10B cells already containing pEBAC140. Electrocompetent cells DH10B (pGETrec, pEBAC140) containing both plasmids were prepared according to standard protocols, except that L-arabinose was included in the growth medium from the first inoculation of the cells. When these cells were electroporated with PCRkan50, nearly 300 recombinants were obtained from two independent electroporations (Table 1, experiments 1, 2). PCR analysis of randomly retrieved recombinant restriction enzyme digestion and 12 with XhoI and HindIII, without any ^Cm r Km ^r recombinants show evidence of rearrangement undesired pEBAC14
0 indicates that the homologous recombination of PCRkan140 has occurred at the correct target site. This was confirmed by DNA sequencing of both homology regions flanking the kanamycin gene. No selection was performed on the pGETrec plasmid on chloramphenicol / kanamycin during propagation of these recombinants, but varying amounts of the multicopy plasmid resulted in a single copy of the pEBAC140 :: kan140 plasmid in different clones. Survived with.

[0081]Example 2 Of 200 kb β-globin clone pEBAC / 148 in DH10B cells
Homologous recombination of PCRkan50 and PCRkan140 into the vector backbone Determine if large single copy BAC clones can be modified using the same method
For this purpose, a 200 kb β-globin clone pE
A modification of BAC / 148 was attempted. pGETrec was converted to DH10B (pEBAC / 1
48) The cells were electroporated. Clos with both plasmids
Electronically using DH10B (pGETrec, pEBAC / 148).
Tent cells were prepared. Induction with L-arabinose for 40 minutes before collecting cells
And made it electrically competent. The element of PCRkan140 in these cells
More than 1000 Cm per electroporation by troproporation ^r Km^rRecombinants were obtained (Table 1, experiments 3, 4). Electrically competent bacteria
If the cells were induced with L-arabinose from the beginning of the culture prior to preparing the cells,
In this case, after electroporation with PCRkan140, Cm^rKm^rset
No transformants were obtained. By L-arabinose inductionrecE,recTAnd
AndgamProlonged overexpression of the gene is toxic to DH10B cells. 20
Cells having 0 kb BAC are longer than cells having the pEBAC140 vector.
It appeared to be more sensitive to the induction induced.

[0082] Twenty independent Cm ^r Km ^r colonies were analyzed by direct PCR in an overnight culture. Primers 140KA and 140KB were replaced with pEBAC / 148 :: kan1.
It was designed to produce a 40 to 1555 bp product. All 20 Cm ^r Km ^r colonies analyzed produced the expected PCR product (Figure 6). The parental pEBAC / 148 clone was used as a control. The control PCR was pEBAC /
The expected fragment from 148 to 393 bp was generated. This fragment was not produced from any of the recombinant clones, but this fragment would have been detected if PCRkan140 integration had occurred anywhere on the bacterial chromosome. Therefore, PCR analysis shows that the recombination event has occurred correctly in any of the 20 independent clones.

DNA was isolated from six of these Cm ^r Km ^r colonies.
Large and variable amounts of multicopy pGETrec plasmids are single copy pEBA
Recovered with C / 148 :: kan140 BAC and prevented interpretation of restriction digestion of BAC. 1 μl of DNA from four clones was re-electroporated into DH10B cells to obtain a number of independent secondary Cm ^r Km ^r colonies from each clone. Isolating DNA from two independent secondary clones of each primary clone,
After digestion with NotI or XhoI, analysis was performed by pulse field gel electrophoresis (FIG. 7). All eight secondary clones contain only large BAC,
The kanamycin resistance marker was shown to be present as an integral part of pEBAC / 148 in these clones. NotI digestion (FIG. 7a) shows that all eight secondary clones release the expected 185 kb β-globin insert,
This indicates that no rough rearrangement has occurred. Also, the size of the bands of the NotI digested vector from all eight clones was the parent clone pEBAC
There was a slight but apparent increase compared to the band of the NotI digested vector at / 148, which corresponded to the expected change in the size of the vector band after incorporation of PCRkan140 into the target site. The same eight clones were further analyzed after restriction digestion with XhoI (FIG. 7b). Except for the approximately 70 kb fragment observed only in the parental pEBAC / 148 clone, all clones were control parental pEBAC / 148 clones.
It showed the same restriction fragments as the EBAC / 148 clone. 7 in 8 secondary clones
The 0 kb fragment appeared to have been cut into two fragments of approximately 30 kb and 40 kb, respectively. Because fragments of 40kb in length present in the parent of pEBAC / 148 clones are moved along with the other fragment of about 40kb, ^Cm r Km parent p is ^r clones
A significantly stronger band was shown at this position compared to the 40 kb band generated by the EBAC / 148 clone. Since there is no XhoI site in the backbone of the parent vector, the 70 kb fragment must be derived from a globin sequence adjacent to the vector backbone. After PCRkan140 has been integrated into the vector backbone by homologous recombination, the 70 kb fragment is cut into two smaller fragments at the single XhoI site present in the kanamycin gene.

These results confirm that the integration of PCRkan140 by homologous recombination occurs precisely at the target site on the backbone of the vector in all four primary Cm ^r Km ^r clones. Furthermore, it is clear that none of these clones showed instability during this procedure, and transient induction of homologous recombination into DH10B cells using the pGETrec plasmid was at least about 200 kDa.
It shows that the risk of undesired rearrangements in BAC clones is minimized up to size b. A more detailed analysis of the homology region used for integration by homologous recombination was performed. pEBAC / 148 :: kan140 clone and pEBA
Sequencing across the region of homology flanking the kanamycin gene in both C140 :: kan50 clones did not show any deviation from the expected sequence.

The PCR product that introduces a short homology region of only 50 bp is JC8679
Or JC9604 be recombined with good efficiency in E. coli strain is shown ^{42, 43.} This allows any fragment of interest to be amplified by synthesizing a 70-80 mer containing a 50 bp homology to the target site (at the 5 'end of the primer) and used directly for targeting by electroporation. This method was performed using PCRkan50 in DH10B (pGETrec, pEB).
AC / 148) Tested with current system by electroporation into cells. The cells were induced with L-arabinose for 40 minutes during preparation. More than 100 Cm ^r Km ^r colonies were obtained from each electroporation (
Table 1, experiments 5, 6). These C using primers 140KF and 140KR
Direct PCR screening of 20 independent overnight cultures from m ^r Km ^r colonies produced the correct 1450bp product.

The use of the 50-mer targeting site reduced the frequency of recombinants by about 10-fold compared to the 140-mer targeting site, but did not alter the accuracy of integration by homologous recombination. Was. No recombinant with the pGETrec plasmid was obtained in DH10B cells without L-arabinose induction, but prolonged induction with L-arabinose is clearly toxic to the cells. Induction for more than 4 hours during preparation of electrically competent cells and recovery after electroporation appeared to significantly impair the viability of the cells, and no recombinants were obtained. On the other hand, only for 10 minutes
-DH10B induced with arabinose (pGETrec, pEBAC / 148)
Electroporation of PCRkan50 into cells produced about 10-fold less Cm ^r Km ^r than after about 40 minutes of induction. Although induction for 40 minutes produced recombinant clones with high efficiency, parameter modulation can be used as a means of modulating recombination efficiency.

Example 3 Construction of pEBAC / 148 The second generation pEBAC140 BAC / PAC cloning vector (FIG. 1) has several properties from the first generation PAC cloning system ²⁴ and BAC cloning system ^{23 and} from the HAEC system ^48. Epstein-Barr virus oriP
And EBNA-1 . The cloning vector also comprises
Includes some additional features, including rare cutter multiple cloning regions.
Preparation of pEBAC / 148 (FIG. 2) was performed from pEBAC140 by retrofitting a 185 kb genomic fragment containing the entire β-globin locus using a procedure based on PAC cloning protocol ⁴⁹ .

Briefly, the RPCI1 human whole genome PAC library ⁴⁹ was screened with PCR primers from the 5 ′ and 3 ′ ends of the β-globin locus. A PAC clone with a 185 kb genomic insert was identified (PA
C148 / β-globin), the entire β-globin locus (about 73 kb) and 5 ′
And both ends of 3 'were shown to contain additional sequences. The 185 kb genomic insert was isolated as a single fragment by NotI digestion and subjected to pulsed field gel electrophoresis (CHEF-DRII, BIO-RAD Labs, Hercules, Calif.), Gelase (Epicentre Technologies, Madison, Wis.). Digested. The pEBAC140 vector was also digested with NotI and purified 18
Before ligating to the 5 kb globin fragment, it was dephosphorylated and gel purified. DH1
After electroporation into OB cells, three independent pEBAC / 14
Eight clones were isolated. Analysis by NotI digestion and pulse field gel electrophoresis showed that all of these clones showed a total of 185 kb β-
Globin genomic fragment. pEBAC / 148 expression of β- globin gene in pEBAC / 148 in stable cell lines that are held in the form of an episome were observed ^50.

Example 4 Construction of pGETrec-Inducible Recombinant Vector The gam gene of bacteriophage λ was used as a primer for gam-F,

Embedded image (SEQ ID NO: 1) and gam-R,

Embedded image (SEQ ID NO: 2) was PCR amplified from plasmid PTP223 ⁴¹ which had been digested with BamHI was used to clone into the BglII site of pBAD24-recET ^43. A clone having the gam gene in the correct orientation with respect to the arabinose promoter was called pGETrec (FIG. 3).

Example 5 L-arabinose induction of recE, recT, and λ gam genes and preparation of electrically competent cells DH10B cells (GIBCO-BRL, gay The overnight culture (Sirsburgh, MD) was diluted 50-fold into 250 ml fresh LB medium containing 100 μg / ml ampicillin and 12.5 μg / ml chloramphenicol. L-arabinose (SIGMA, St. Louis, Mo.) was added to a final concentration of 0.2% (w / v) when the OD600 of the cells reached 0.40-0.42. Electrocompetent cells were prepared by harvesting at OD ₆₀₀ 0.55-0.60 using the protocol recommended by the manufacturer. The final cell pellet was resuspended in a total volume of 400 to 500 μl of 10% glycerin. The cells were aliquoted into pre-cooled 1.5 ml microcentrifuge tubes (40 μl / tube) and frozen in a dry ice-ethanol bath. The frozen cells were stored at -70 ° C.

[0091] The kanamycin gene from Preparation vector PCYPAC2 ⁴⁹ Example 6 targeting DNA fragment PCRkan50 and PCRkan140, was amplified using the following primers: Kan50F

Embedded image (SEQ ID NO: 3) and kan50R

Embedded image (SEQ ID NO: 4). The first 50 nucleotides of each primer sequence (underlined) correspond to sequences in the upstream and downstream strands of the pEBAC140 vector bordering the unique Bst11071 site, while the last 20 nucleotides of each primer are pCYPAC2 It corresponds to the sequence flanking the kanamycin gene in the vector. The PCR kan50 of the PCR product is 1262 bp long, and the kanamycin gene and promoter region and pEBAC1
Includes 50-mer sequences at both ends for targeting integration into the Bst11071 site on the 40 vector (FIG. 4). Using this PCR product, pEBAC140 :: kan50 was generated by targeting the integration of PCRkan50 into pEBAC140 in E. coli JC8679 cells. P
CRkan140 is 1450 bp long and has a primer 140KF

Embedded image (SEQ ID NO: 5) and 140KR

Embedded image PC from pEBAC140 :: kan50 by using (SEQ ID NO: 6)
Generated by R. PCRkan140 has homologous targeting sequences of 145 bp and 143 bp at the 5 'and 3' ends of the kanamycin gene, respectively. The PCR products were purified by gel electrophoresis, recovered with the QIAquick gel extraction kit (Qiagen, GmbH, Hilden, Germany) and then quantified.

Example 7 Production of Recombinants The overall procedure for producing recombinants is outlined (FIG. 5). 40 μl of the frozen stock of electrically competent cells in a microcentrifuge tube is thawed on ice and
ng of PCRkan50 or PCRkan140 was added to the cells in each tube. DN
The mixture of A and competent cells was quickly transferred to a pre-chilled 0.2 cm electrode gap cuvette (BIO-RAD Labs, Hercules, CA) and electroporated using a BIO-RAD Gene Pulser instrument. did. Electroporation conditions were 2.5 kV, 200 Ω, 25 μF. Immediately after the electroporation, 1 ml of the LB medium was added to the cuvette, and the cells were 17 × 1
Transfer to a 00 mm FALCON polystyrene tube (BECKTON-DICKINSON Labware, Lincoln Park, NJ). Tube was shaken at 37 ° C (220r
pm) for 1.5 hours. Cells from each electroporation were spread on LB plates containing 12.5 μg / ml chloramphenicol and 35 μg / ml kanamycin to identify recombinants. The measurement of the electroporation efficiency of each batch of competent cells was performed using 10 ng of pZeoSV.
2 (Invitrogen, Carlsbad, CA) by independent electroporation and low salt LB containing 25 μg / ml Zeocin (pH
7.5) This was performed by plating serial dilutions of the electroporation mixture on a plate. Measurement of the total number of cells surviving after electroporation was performed using DH10B (pGETrec, pEBAC140) or DH10B (
Serial dilutions of electroporated competent cells (without addition of DNA) of pGETrec, pEBAC / 148) were performed on LB plates containing 100 μg / ml ampicillin and 1.25 μg / ml chloramphenicol. Performed by plating. Percent recombination was measured as the percentage of colonies resistant to kanamycin and chloramphenicol per colony that survived electroporation.

EXAMPLE 8 Analysis of Recombinants DNA from chloramphenicol and kanamycin resistant colonies was purified using a modified alkaline lysis miniprep method ⁴⁹ at 12.5 μg / ml chloramphenicol and 35 μg / ml. 2 grown in LB medium containing kanamycin
ml of overnight culture. BAC D digested with NotI or XhoI
NA was analyzed by pulsed-field gel electrophoresis in 1% (w / v) agarose gel (SeaKem, FMC Bioproducts, Rockland, Me.) In 0.5 × TBE buffer at 6 V / cm, 14 ° C. . Low Range and Midrange IPFG markers (NEB, Inc., Beverly, Mass.) Were used as standards. PCR analysis of the recombinant clones was performed using AmpliTaq DNA
Overnight cultures were performed directly using a polymerase kit (Perkin Elmer Corp., Norwalk, Connecticut). Recombinant (2 ml grown in the presence of 12.5 μg / ml chloramphenicol and 35 μg / ml kanamycin
1 μl aliquot of the parent clone (grown in the presence of only 12.5 μg / ml chloramphenicol) was added directly to the PCR reaction. p
The recombinant product of EBAC140 and PCRkan50 was converted to pEBAC140 :: ka
Indicated as n50. Similarly, the recombinant product of pEBAC / 148 and PCRkan50 or PCRkan140 was designated as pEBAC / 148 :: kan, respectively.
50 and pEBAC / 148 :: kan140. pEBAC1
The PCR primers used to verify 40 :: kan140 and pEBAC / 148 :: kan140 were 140KA,

Embedded image (SEQ ID NO: 7) and 140 KB,

Embedded image (SEQ ID NO: 8). pEBAC / 148 :: kan50 and pEBAC
The PCR primer used to confirm both 140 :: kan50 was 140K
F and 140 KR (FIG. 4). PCR products were separated on a 1.5% (w / v) agarose gel. The PCR products were sequenced at the recombinant junctions using a Thermosequenase radiolabeled terminator cycle sequencing kit (American Life Sciences, Inc., Cleveland, Ohio).

[0094] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described herein. It should be understood that the present invention covers all such changes and modifications. The present invention also includes all of the steps, features, compositions and complexes referred to or indicated herein individually or as a whole, and combinations of two or more of the steps or features.

References

[Table 1]

[0096]

[Table 2]

[0097]

[Table 3]

[0098]

[Table 4]

[0099]

[Table 5]

[0100]

[Table 6]

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the pEBAC140 vector map, showing its main features. This vector is based on the backbone of the F plasmid pBeloBAC11 ²³ . The hygromycin and thymidine kinase genes allow for selection in eukaryotic cells, while the oriP and EBNA-1 genes from Epstein Barr virus facilitate episomal maintenance. This vector, la
It contains a unique rare cutter multiple cloning site inserted in-frame into the cZ gene, by either blue / white selection of recombinant and many different cutters flanking the BamHI cloning site. It allows for the recovery of genomic inserts. The modified pUC19-Xho is inserted into the XhoI site at a later stage in the vector, allowing multiple copies and facilitating cloning applications.

FIG. 2 is a schematic diagram of pEBAC / 148 obtained by inserting a 185 kb genomic fragment from a PAC clone containing the complete β-globin genomic region into the NotI site of the pEBAC140 vector. The location in this clone targeting the integration of the kanamycin resistance gene by homologous recombination and the location in the first pEBAC140 vector are shown.

FIG. 3 is a schematic diagram of a map showing main features of the pGETrec plasmid.
is there. Bacteriophage λgamThe PCR product containing the gene was converted into pBAD24-
recET plasmid⁴⁵To generate pGETrec. therefore, gam The gene is arabinose with the recE and recT genes.
Under the control of the motor.

FIG. 4 is a schematic diagram of the oligonucleotide primer used. PCR kan50 (1262 bp) was generated by amplifying the kanamycin gene from pCYPAC2 using primers kan50F and kan50R. Primers kan50F and kan50R also contain a 50-mer targeting region homologous to the sequence flanking the unique Bst1107I site on the vector target sequence. PCRkan50 into pEBAC140 vector
After homologous recombination, the PCRkan140 vector was generated using primers KF and KR. The expected size of the PCR product using primers KF / KR was 288 bp and 1 in the absence and presence of the kanamycin gene, respectively.
450 bp. The expected size of the corresponding PCR product containing primers KA / KB is 393 bp and 1555 bp.

[FIG. 5] A homologous recombination of PCR fragment into BAC / P in DH10B cells.
FIG. 2 is a schematic diagram showing the “GET recombination” method for incorporation into AC. The PCR primers each have a specific sequence at the 3 'end for amplifying a DNA fragment with a selectable marker, along with a 5' to a targeting site in PAC or BAC.
It is designed to include a 0-mer homology region. Additional sequences or markers may also be included. The PCR product generated by these primers is electroporated into electrocompetent DH10B cells containing both the BAC or PAC clone of interest and the pGETrec plasmid, and the electrocompetent cells are subjected to electroporation. Is transiently induced with 0.2% L-arabinose during preparation. Recombination between the homology region of the PCR fragment and the BAC or PAC clone allows for integration of the PCR fragment. Modified BAC or PAC clones were used for miniprep DNA isolation and DH10B
It is purified from pGETrec by electroporation into cells or by streaking with antibiotics in the medium.

FIG. 6. 20 independent Cs with pEBAC / 148 :: kan140
It is a photographic display of PCR analysis of m ^r Km ^r colonies. One μl of an overnight culture grown from each Cm ^r Km ^r colony was added to a PCR reaction using PCR primers KF and KR. PCR products were separated using a 1.5% (w / v) agarose gel. Lanes: 1 and 24, 1-kb DNA ladder standard; lanes 2-2
1,20 independent Cm ^r Km ^r colonies; lane 22, parental pEBAC / 148
Clone; lane 23, negative control (no cells).

FIG. 7. Four Cm ^r Km ^r colonies (A-D) randomly picked after incorporation of PCRkan140 into pEBAC / 148 by homologous recombination
5 is a photograph display of the PFGE analysis of FIG. DNA from each clone was again electroporated into DH10B cells. The second colony of the two copies was DN
A used for miniprep extraction and restriction enzyme digestion with NotI and XhoI.
(A) NotI digestion: M, low range PFG marker; lanes 1-8, 2 copies of p
EBAC / 148 :: kan140 clone; P, parental pEBAC / 148 clone. PFGE conditions: 6 V / cm, switch time 0.1 to 25.0 seconds, 14 ° C,
17 hours. (B) XhoI digestion: M, low range PFG marker; lanes 1-8, 2 copies of p
EBAC / 148 :: kan140 clone; P, parental pEBAC / 148 clone; M2, medium range IPFG marker. PFGE conditions: 6 V / cm
, Switch time 0.1-8.0 seconds, 14 ° C, 18 hours.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // A61K 31/7088 C12N 15/00 ZNAA 4C086 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, C , CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Faculty of Research Science and Technology F-term (reference) 2G045 BB20 DA12 DA13 DA14 FB02 4B024 AA01 AA11 AA20 BA80 CA03 DA06 EA04 HA17 4B065 AA26X AA90Y AA93Y AB01 AC14 BA02 CA44 CA60 4C057 AMM4 C086 AA01 AA03 EA16 MA01 MA04 NA20

Claims (55)

[Claims] 1. A method for facilitating homologous recombination between at least two nucleotide sequences, said recBCD being under conditions sufficient to facilitate homologous recombination of said at least two nucleotide sequences. Culturing a host cell capable of expressing a nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or a derivative thereof to induce recombination between said at least two nucleotide sequences in said host cell. Wherein the expression of said exonuclease, recombinant protein and recBCD inhibitor is modulatable. 2. The method of claim 1, wherein said at least two nucleotide sequences are a cyclic nucleotide sequence and a linear nucleotide sequence. 3. The method of claim 2, wherein said cyclic nucleotide sequence comprises an F plasmid portion. 4. The method according to claim 3, wherein said F plasmid portion is BAC. 5. The method of claim 3, wherein said F plasmid portion is PAC. 6. The method according to claim 1, wherein the exonuclease is recE or a functional derivative thereof, and the recombinant protein is recT or a functional derivative thereof.
6. The method according to any one of claims 1 to 5. 7. The method of claim 6, wherein said recBCD inhibitor is gam, P22Abc or SSB or a functional derivative thereof. 8. The method of claim 7, wherein said recBCD inhibitor is gam or a functional derivative thereof. 9. The method of claim 8, wherein said nucleic acid molecule encoding recE, recT and gam is operably linked to an inducible promoter. 10. The method of claim 9, wherein said nucleic acid molecules encoding recE, recT and gam are operably linked to a common inducible promoter. 11. The method according to claim 9, wherein the promoter is L-arabinose. 12. The method of claim 11, wherein said nucleic acid molecule operably linked to a common L-arabinose promoter corresponds to the expression plasmid pGETrec. 13. The method according to claim 1, wherein the host cell is Escherichia coli. 14. The method of claim 13, wherein said E. coli expresses recBCD. 15. The method according to claim 14, wherein said E. coli is strain RR1, DM1 or DH10B. 16. The method of claim 15, wherein said strain is DH10B. 17. A method for facilitating homologous recombination of at least two nucleotide sequences in a host cell, comprising the steps of: (i) transforming the host cell, wherein the transformed host cell comprises , A first nucleotide sequence encoding an exonuclease, a recombinant protein and a recBCD inhibitor or a functional derivative thereof, and the at least two nucleotide sequences, wherein the exonuclease, the recombinant protein, and the recBC
Expression of the gene encoding the D inhibitor can be modulated, and (ii) culturing the transformed host cell for a time and under conditions sufficient for expression of the first nucleotide sequence to occur; Wherein the expression product of the first nucleotide sequence facilitates homologous recombination of the second nucleotide sequence with the third nucleotide sequence. 18. The method of claim 17, wherein said at least two nucleotide sequences are a cyclic nucleotide sequence and a linear nucleotide sequence. 19. The method of claim 18, wherein said cyclic nucleotide sequence comprises an F plasmid portion. 20. The method of claim 19, wherein said F plasmid portion is BAC. 21. The method of claim 19, wherein said F plasmid portion is PAC. 22. The method according to claim 17, wherein the exonuclease is recE or a functional derivative thereof, and the recombinant protein is recT or a functional derivative thereof. 23. The method of claim 22, wherein said recBCD inhibitor is gam, P22Abc or SSB or a functional derivative thereof. 24. The method of claim 23, wherein said recBCD inhibitor is gam or a functional derivative thereof. 25. The method of claim 24, wherein said nucleic acid molecule encoding recE, recT and gam is operably linked to an inducible promoter. 26. The method of claim 25, wherein said nucleic acid molecules encoding recE, recT and gam are operably linked to a common inducible promoter. 27. The method according to claim 25, wherein the promoter is L-arabinose. 28. The method of claim 27, wherein said nucleic acid molecule operably linked to a common L-arabinose promoter corresponds to the expression plasmid pGETrec. 29. The method according to claim 17, wherein said host cell is Escherichia coli. 30. The method of claim 29, wherein said E. coli expresses recBCD. 31. The method of claim 30, wherein said E. coli is strain RR1, DM1 or DH10B. 32. The method of claim 31, wherein said strain is DH10B. 33. A method of producing a microorganism useful for facilitating homologous recombination between at least two nucleotide sequences, comprising recBCD, a modulatable level of an exonuclease, a recombinant protein, and a recBCD inhibitor. And genetically engineering a host cell to allow expression of the at least two other nucleotide sequences. 34. The method of claim 33, wherein said at least two nucleotide sequences are a cyclic nucleotide sequence and a linear nucleotide sequence. 35. The method of claim 34, wherein said cyclic nucleotide sequence comprises an F plasmid portion. 36. The method of claim 35, wherein said F plasmid portion is BAC. 37. The method of claim 35, wherein said F plasmid portion is PAC. 38. The method according to claim 33, wherein said exonuclease is recE or a functional derivative thereof, and said recombinant protein is recT or a functional derivative thereof. 39. The method of claim 38, wherein said recBCD inhibitor is gam, P22Abc or SSB or a functional derivative thereof. 40. The method of claim 39, wherein said recBCD inhibitor is gam or a functional derivative thereof. 41. The method of claim 40, wherein said nucleic acid molecule encoding recE, recT and gam is operably linked to an inducible promoter. 42. The method of claim 41, wherein said nucleic acid molecules encoding recE, recT and gam are operably linked to a common inducible promoter. 43. The method according to claim 41 or 42, wherein the promoter is L-arabinose. 44. The method of claim 43, wherein said nucleic acid molecule operably linked to a common L-arabinose promoter corresponds to the expression plasmid pGETrec. 45. The method according to claim 33, wherein said host cell is Escherichia coli. 46. The method of claim 45, wherein said E. coli expresses recBCD. 47. The method of claim 45, wherein said E. coli is strain RR1, DM1 or DH10B. 48. The method of claim 47, wherein said strain is DH10B. 49. A cell capable of facilitating homologous recombination between at least two nucleotide sequences, comprising: an exonuclease, a nucleotide sequence encoding a recombinant protein and a gene encoding a recBCD inhibitor; and A cell comprising a nucleotide sequence, wherein the expression of said exonuclease, recombinant protein and recBCD inhibitor is modulatable. 50. The cell according to claim 49, wherein said homologous recombination is performed according to the method according to any one of claims 1 to 32. 51. A nucleic acid molecule that has undergone homologous recombination according to the method of claim 1. Description: 52. Use of a nucleic acid molecule according to claim 51 in the manufacture of a medicament for treating a disease in a mammal. 53. Use of a nucleic acid molecule according to claim 51 in diagnosing a condition in a mammal. 54. A pharmaceutical composition comprising a nucleic acid molecule according to claim 51 together with one or more pharmacologically acceptable carriers and / or diluents. 55. A kit for facilitating homologous recombination of at least two nucleotide sequences in a host cell, wherein the kit encodes an exonuclease, a recombinant protein, a recBCD inhibitor or a functional derivative thereof. A kit comprising the above nucleotide sequence and a compartment adapted to contain reagents useful for facilitating homologous recombination.