US20090170727A1 - Methods for dynamic vector assembly of dna cloning vector plasmids - Google Patents

Methods for dynamic vector assembly of dna cloning vector plasmids Download PDF

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US20090170727A1
US20090170727A1 US11/569,335 US56933505A US2009170727A1 US 20090170727 A1 US20090170727 A1 US 20090170727A1 US 56933505 A US56933505 A US 56933505A US 2009170727 A1 US2009170727 A1 US 2009170727A1
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insert
promoter
expression
cleaved
regulatory
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Thomas D. Reed
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Precigen Inc
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Intrexon Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates in general to the field of cloning vector plasmids, and in particular to methods for rapidly assembling DNA constructs or transgenes with cloning vector plasmids.
  • the foundation of molecular biology is recombinant DNA technology, which can here be summarized as the modification and propagation of nucleic acids for the purpose of studying the structure and function of the nucleic acids and their protein products.
  • genes, gene regulatory regions, subsets of genes, and indeed entire chromosomes in which they are contained are all comprised of double-stranded anti-parallel sequences of the nucleotides adenine, thymine, guanine and cytosine, identified conventionally by the initials A, T, G, and C, respectively.
  • DNA sequences, as well as cDNA sequences, which are double stranded DNA copies derived from mRNA (messenger RNA) molecules can be cleaved into distinct fragments, isolated, and inserted into a vector such as a bacterial plasmid to study the gene products.
  • a plasmid is an extra-chromosomal piece of DNA that was originally derived from bacteria, and can be manipulated and reintroduced into a host bacterium for the purpose of study or production of a gene product.
  • the DNA of a plasmid is similar to all chromosomal DNA, in that it is composed of the same A, T, G, and C nucleotides encoding genes and gene regulatory regions, however, it is a relatively small molecule comprised of less than approximately 30,000 base-pairs, or 30 kilobases (kb).
  • the nucleotide base pairs of a double-stranded plasmid form a continuous circular molecule, also distinguishing plasmid DNA from that of chromosomal DNA.
  • Plasmids enhance the rapid exchange of genetic material between bacterial organisms and allow rapid adaptation to changes in environment, such as temperature, food supply, or other challenges. Any plasmid acquired must express a gene or genes that contribute to the survival of the host or else it will be destroyed or discarded by the organism, since the maintenance of unnecessary plasmids would be a wasteful use of resources.
  • a clonal population of cells contains identical genetic material, including any plasmids it might harbor.
  • Use of a cloning vector plasmid with a DNA insert in such a clonal population of host cells will amplify the amount of the DNA of interest available. The DNA so cloned may then be isolated and recovered for subsequent manipulation in the steps required for building a DNA construct.
  • cloning vector plasmids are useful tools in the study of gene function, providing the ability to rapidly produce large amounts of the DNA insert of interest.
  • a particularly useful plasmid-born gene that can be acquired by a host is one that would confer antibiotic resistance.
  • antibiotic resistance genes are exploited as positive or negative selection elements to preferentially enhance the culture and amplification of the desired plasmid over that of other plasmids.
  • a plasmid In order to be maintained by a host bacterium, a plasmid must also contain a segment of sequences that direct the host to duplicate the plasmid. Sequences known as the origin of replication (ORI) element direct the host to use its cellular enzymes to make copies of the plasmid. When such a bacterium divides, the daughter cells will each retain a copy or copies of any such plasmid. Certain strains of E. coli bacteria have been derived to maximize this duplication, producing upwards of 300 copies per bacterium. In this manner, the cultivation of a desired plasmid can be enhanced.
  • ORI origin of replication
  • MCS multiple cloning site
  • the so-called recognition sequences of a restriction endonuclease (RE) site encoded in the DNA molecule comprise double-stranded palindromic sequences.
  • RE enzymes as few as 4-6 nucleotides are sufficient to provide a recognition site, while some RE enzymes require a sequence of 8 or more nucleotides.
  • the RE enzyme EcoR1 recognizes the double-stranded hexanucleotide sequence: 5′ G-A-A-T-T-C 3′ , wherein 5′ indicates the end of the molecule known by convention as the “upstream” end, and 3′ likewise indicates the “downstream” end.
  • the complementary strand of the recognition sequence would be its anti-parallel strand, 3′ G-A-A-T-T-C- 5′ . Since every endonuclease site is a double-stranded sequence of nucleotides, a recognition site of 6 nucleotides is, in fact, 6 base pairs (bp). Thus the double stranded recognition site can be represented within the larger double-stranded molecule in which it occurs as:
  • EcoR1 does not cleave exactly at the axis of dyad symmetry, but at positions four nucleotides apart in the two DNA strands between the nucleotides indicated by a “/”:
  • a further general principle to consider when building recombinant DNA molecules is that all endonuclease sites occurring within a molecule will be cut with a particular RE enzyme, not just the site of interest. The larger a DNA molecule, the more likely it is that any endonuclease site will reoccur.
  • a tetranucleotide site will occur, on the average, once every 4 4 (i.e., 256) nucleotides or bp, whereas a hexanucleotide site will occur once every 4 6 (i.e., 4096) nucleotides or bp, and octanucleotide sites will occur once every 4 8 (i.e., 114,688) nucleotides or bp.
  • 4 4 i.e., 256
  • a hexanucleotide site will occur once every 4 6 (i.e., 4096) nucleotides or bp
  • octanucleotide sites will occur once every 4 8 (i.e., 114,688) nucleotides or bp.
  • Frequently-occurring endonuclease enzyme sites are herein referred to as common sites, and the endonucleases that cleave these sites are referred to as common endonuclease enzymes.
  • Restriction enzymes with cognate restriction sites greater than 6 bp are referred to as rare restriction enzymes, and their cognate restriction sites as rare restriction sites.
  • rare restriction enzymes Restriction enzymes with cognate restriction sites greater than 6 bp
  • rare restriction sites are also referred to as rare.
  • the designations “rare” and common” do not refer to the relative abundance or availability of any particular restriction enzyme, but rather to the frequency of occurrence of the sequence of nucleotides that make up its cognate recognition site within any DNA molecule or isolated fragment of a DNA molecule, or any gene or its DNA sequence.
  • HE enzymes have large, non-palindromic asymmetric recognition sites (12-40 base pairs). HE recognition sites are extremely rare. For example, the HE known as I-SceI has an 18 bp recognition site, (5′ . . . TAGGGATAACAGGGTAAT . . . 3′), predicted to occur only once in every 7 ⁇ 10 10 bp of random sequence. This rate of occurrence is equivalent to only one site in 20 mammalian-sized genomes. The rare nature of HE recognition sites greatly increases the likelihood that a genetic engineer can cut a final transgene product without disrupting the integrity of the transgene if HE recognition sites were included in appropriate locations in a cloning vector plasmid.
  • a DNA molecule from any source organism will be cut in identical fashion by an endonuclease enzyme, foreign pieces of DNA from any species can be cut with an endonuclease enzyme, inserted into a bacterial plasmid vector that was cleaved with the same endonuclease enzyme, and amplified in a suitable host cell.
  • an endonuclease enzyme e.g. EcoR1
  • the desired fragment with EcoR1 ends can be isolated and mixed with a plasmid that was also cut with EcoR1 in what is commonly known as a ligation mixture. Under the appropriate conditions in the ligation mixture, some of the isolated human gene fragments will match up with the ends of the plasmid molecules.
  • Transgenes Recombinant DNA technology is frequently embodied in the generation of so-called “transgenes”.
  • Transgenes frequently comprise a variety of genetic materials that are derived from one or more donor organisms and introduced into a host organism.
  • a transgene is constructed using a cloning vector as the starting point or “backbone” of the project, and a series of complex cloning steps are planned to assemble the final product within that vector.
  • Elements of a transgene comprising nucleotide sequences, include, but are not limited to 1) regulatory promoter and/or enhancer elements, 2) a gene that will be expressed as a mRNA molecule, 3) DNA elements that provide mRNA message stabilization, 4) nucleotide sequences mimicking mammalian intronic gene regions, and 5) signals for mRNA processing such as the poly-A tail added to the end of naturally-occurring mRNAs.
  • an experimental design may require addition of localization signal to provide for transport of the gene product to a particular subcellular location.
  • Each of the elements of a transgene can be derived as a fragment of a larger DNA molecule that is cut from a donor genome, or, in some cases, synthesized in a laboratory. While the present invention employs endonucleases for the methods claimed herein, it is known that each of the smaller elements comprising, for example, the inserts or modules which are used in the methods herein, can be created by de novo synthesis, recombineering, and/or PCR terminator overhang cloning.
  • One such method of synthesis of the component elements of a transgene includes the method disclosed by Jarrell et al. in U.S. Pat. No. 6,358,712, which is incorporated herein by reference in its entirety.
  • Jarrell discloses a method for “welding” elements of a transgene together
  • the methods of the present invention disclose a way to “unweld” and re-assemble the elements once they have been assembled.
  • each piece is assembled with the others in a precise order and 5′-3′ orientation into a cloning vector plasmid.
  • the promoter of any gene may be isolated as a DNA fragment and placed within a synthetic molecule, such as a plasmid, to direct the expression of a desired gene, assuming that the necessary conditions for stimulation of the promoter of interest can be provided.
  • the promoter sequences of the insulin gene may be isolated, placed in a cloning vector plasmid along with a reporter gene, and used to study the conditions required for expression of the insulin gene in an appropriate cell type.
  • the insulin gene promoter may be joined with the protein coding-sequence of any gene of interest in a cloning vector plasmid, and used to drive expression of the gene of interest in insulin-expressing cells, assuming that all necessary elements are present within the DNA transgene so constructed.
  • a reporter gene is a particularly useful component of some types of transgenes.
  • a reporter gene comprises nucleotide sequences encoding a protein that will be expressed under the direction of a particular promoter of interest to which it is linked in a transgene, providing a measurable biochemical response of the promoter activity.
  • a reporter gene is typically easy to detect or measure against the background of endogenous cellular proteins. Commonly used reporter genes include but are not limited to LacZ, green fluorescent protein, and luciferase, and other reporter genes, many of which are well known to those skilled in the art.
  • Introns which are non-coding regions within mammalian genes, are not found in bacterial genomes, but are required for proper formation of mRNA molecules in mammalian cells. Therefore, any DNA construct for use in mammalian systems must have at least one intron. Introns may be isolated from any mammalian gene and inserted into a DNA construct, along with the appropriate splicing signals that allow mammalian cells to excise the intron and splice the remaining mRNA ends together.
  • An mRNA stabilization element is a sequence of DNA that is recognized by binding proteins that protect some mRNAs from degradation. Inclusion of an mRNA stabilization element will frequently enhance the level of gene expression from that mRNA in some mammalian cell types, and so can be useful in some DNA constructs or transgenes.
  • An mRNA stabilization element can be isolated from naturally occurring DNA or RNA, or synthetically produced for inclusion in a DNA construct.
  • a localization signal is a sequence of DNA that encodes a protein signal for subcellular routing of a protein of interest.
  • a nuclear localization signal will direct a protein to the nucleus; a plasma membrane localization signal will direct it to the plasma membrane, etc.
  • a localization signal may be incorporated into a DNA construct to promote the translocation of its protein product to the desired subcellular location.
  • a tag sequence may be encoded in a DNA construct so that the protein product will have a unique region attached.
  • This unique region serves as a protein tag that can distinguish it from its endogenous counterpart.
  • it can serve as an identifier that may be detected by a wide variety of techniques well known in the art, including, but not limited to, RT-PCR, immunohistochemistry, or in situ hybridization.
  • transgene design and construction consumes significant amounts of time and energy for several reasons, including the following:
  • endonuclease enzymes There is a wide variety of endonuclease enzymes available that will generate an array of termini, however most of these are not compatible with each other. Many endonuclease enzymes, such as EcoR1, generate DNA fragments with protruding 5′ cohesive termini or “tails”; others (e.g., Pst1) generate fragments with 3′ protruding tails, whereas still others (e.g., Bal1) cleave at the axis of symmetry to produce blunt-ended fragments. Some of these will be compatible with the termini formed by cleavage with other endonuclease enzymes, but the majority of useful ones will not. The termini that can be generated with each DNA fragment isolation must be carefully considered in designing a DNA construct.
  • DNA fragments needed for assembly of a DNA construct or transgene must first be isolated from their source genomes, placed into plasmid cloning vectors, and amplified to obtain useful quantities. The step can be performed using any number of commercially-available or individually altered cloning vectors. Each of the different commercially available cloning vector plasmids were, for the most part, developed independently, and thus contain different sequences and endonuclease sites for the DNA fragments of genes or genetic elements of interest. Genes must therefore be individually tailored to adapt to each of these vectors as needed for any given set of experiments. The same DNA fragments frequently will need to be altered further for subsequent experiments or cloning into other combinations for new DNA constructs or transgenes. Since each DNA construct or transgene is custom made for a particular application with no thought or knowledge of how it will be used next, it frequently must be “retrofitted” for subsequent applications.
  • DNA sequence of any given gene or genetic element varies and can contain internal endonuclease sites that make it incompatible with currently available vectors, thereby complicating manipulation. This is especially true when assembling several DNA fragments into a single DNA construct or transgene.
  • the present invention provides a method of rapidly assembling DNA constructs or transgenes by using cloning vector plasmids.
  • the invention also provides a method that incorporates multiple DNA fragments, also known as both “inserts” or “modules”, such as one each of a Promoter, Expression, and 3′ Regulatory nucleotide sequence, into a cloning vector plasmid in a single step, rather than having to introduce each insert in a sequential manner.
  • a method is called “Dynamic Vector Assembly” herein.
  • the present invention provides a method for constructing a transgene, comprising the steps of providing a cloning vector plasmid with a backbone able to accept a sequential arrangement of inserts, providing at least a first insert and a second insert to be included in the transgene, and transferring both the first insert and the second insert into the backbone in a single reaction.
  • the invention provides a method for making a transgene, comprising the steps of: providing a cloning vector plasmid comprising first and second docking points; introducing first nucleotide sequences to be included in the transgene into a first shuttle vector; introducing second nucleotide sequences to be included in the transgene into a second shuttle vector; and transferring simultaneously the first nucleotide sequences and the second nucleotide sequences from the shuttle vectors to the cloning vector plasmid, between the first and second docking points.
  • the invention also provides a method for making a transgene, comprising the steps of: providing a cloning vector plasmid comprising first and second docking points; introducing Promoter nucleotide sequences to be included in the transgene into a Promoter shuttle vector; introducing Expression nucleotide sequences to be included in the transgene into an Expression shuttle vector; introducing Regulatory nucleotide sequences to be included in the transgene into a Regulatory shuttle vector; and transferring simultaneously the Promoter, Expression and Regulatory nucleotide sequences from the Promoter, Expression and Regulatory shuttle vectors to the cloning vector plasmid, between the first and second docking points.
  • the invention provides a method for simultaneously synthesizing an array of transgenes, comprising the steps of: providing a primary cloning vector plasmid comprising a first and a second docking point; introducing at least one Promoter nucleotide sequence to be included in the transgene into a corresponding Promoter shuttle vector; introducing at least one Expression nucleotide sequence to be included in the transgene into a corresponding Expression shuttle vector; introducing at least one Regulatory nucleotide sequence to be included in the transgene into a corresponding Regulatory shuttle vector; and transferring simultaneously the Promoter, Expression and Regulatory nucleotide sequences from the Promoter, Expression and Regulatory shuttle vectors to the cloning vector plasmid, between the first and second docking points, wherein at least two combinations of one Promoter module, one Expression module, and one Regulatory module are transferred into two distinct primary cloning vector molecules.
  • the invention provides a method for making a modular cloning vector plasmid for the synthesis of a transgene or other complicated DNA construct, the method comprising the steps of: providing the cloning vector plasmid comprising a backbone, the backbone comprising first and second docking points, each docking point being fixed within the backbone and comprising at least one non-variable rare endonuclease site for an endonuclease enzyme; cleaving the first docking point with a first endonuclease enzyme corresponding to the at least one non-variable rare restriction site of the first docking point, leaving the cleaved first docking point with a 3′ end; cleaving the second docking point with a second nuclease enzyme corresponding to the at least one non-variable rare endonuclease site of the second docking point, leaving the cleaved second docking point with a 5′ end; providing at least a first and a second insert,
  • the invention provides a method for synthesizing a transgene or other complicated DNA construct, comprising the steps of: providing a primary cloning vector plasmid comprising a backbone, the backbone comprising at least a first docking point and a second docking point, each docking point being fixed within the backbone and comprising at least one rare restriction site for a non-variable rare restriction enzyme; cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the rare restriction sites of the first docking point, leaving the cleaved backbone with a 3′ end; cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the restriction sites of the second docking point, leaving the cleaved backbone with a 5′ end, providing a Promoter insert into which a Promoter sequence of interest, a 5′ end that is compatible to the 3′ end of the first docking point, and a 3′ end; providing an Expression insert comprising
  • the invention provides a method for simultaneously synthesizing an array of transgenes or other complicated DNA constructs, comprising the steps of: providing at least one primary cloning vector plasmid comprising a backbone into which inserts having a 5′ end, a nucleotide sequence of interest and a 3′ end can be inserted, the backbone operable to accept a sequential arrangement of Promoter, Expression, and Regulatory inserts and comprising at least a first and a second docking point, each docking point being fixed within the backbone and comprising at least one restriction site for a non-variable rare restriction enzyme; cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the restriction sites of the first docking point; cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the restriction sites of the second docking point; providing at least one Promoter insert into which a Promoter nucleotide sequence has been inserted, the 5′ end of
  • FIG. 1 is a linear map of the module concept of the invention showing a Shuttle vector that is insertable into a PE3 docking station, which is insertable into a Primary docking station.
  • FIG. 2 is an illustration depicting assembly of a backbone vector enabled by the relationships between restriction sites within shuttle vectors such as Promoter, Expression and 3′ Regulatory modules, and the docking points on a primary cloning vector plasmid.
  • FIG. 3 is an illustration depicting assembled backbone vector of FIG. 2 .
  • CMD chromatin modification domain
  • cloning refers to the process of ligating a DNA molecule into a plasmid and transferring it an appropriate host cell for duplication during propagation of the host.
  • cloning vector and “cloning vector plasmid” are used interchangeably to refer to a circular DNA molecule minimally containing an Origin of Replication, a means for positive selection of host cells harboring the plasmid such as an antibiotic-resistance gene; and a multiple cloning site.
  • the term “common” in relation to endonuclease sites refers to any endonuclease site that occurs relatively frequently within a genome.
  • the phrase “compatible to” refers a terminus or end, either 5′ or 3′, of a strand of DNA which can form hydrogen bonds with any other complementary termini either cleaved with the same restriction enzyme or created by some other method. Since any DNA that contains a specific recognition sequence for a restriction enzyme will be cut in the same manner as any other DNA containing the same sequence, those cleaved ends will be complementary and thus compatible. Therefore, the ends of any DNA molecules cut with the same restriction enzyme “match” each other in the way adjacent pieces of a jigsaw puzzle “match”, and can be enzymatically linked together. Compatible ends will form a recognition site for a particular restriction enzyme when combined together.
  • de novo synthesis refers to the process of synthesizing double-stranded DNA molecules of any length by linking complementary single-stranded DNA molecules compatible overhangs that represent subsequences of the total desired DNA molecule.
  • DNA construct refers to a DNA molecule synthesized by consecutive cloning steps within a cloning vector plasmid, and is commonly used to direct gene expression in any appropriate cell host such as cultured cells in vitro, or a transgenic mouse in vivo.
  • a transgene used to make such a mouse can also be referred to as a DNA construct, especially during the period of time when the transgene is being designed and synthesized.
  • DNA fragment refers to any isolated molecule of DNA, including but not limited to a protein-coding sequence, reporter gene, promoter, enhancer, intron, exon, poly-A tail, multiple cloning site, nuclear localization signal, or mRNA stabilization signal, or any other naturally occurring or synthetic DNA molecule, or any portion thereof.
  • a DNA fragment may be completely of synthetic origin, produced in vitro.
  • a DNA fragment may comprise any combination of isolated naturally occurring and/or synthetic fragments.
  • the term “Docking Plasmid” refers to a specialized cloning vector plasmid used in the invention to assemble DNA fragments into a DNA construct.
  • the terms “endonuclease” or “endonuclease enzyme” refers to a member or members of a classification of catalytic molecules that bind a recognition site encoded in a DNA molecule and cleave the DNA molecule at a precise location within or near the sequence.
  • cognate recognition site As used herein, the terms “endonuclease recognition site”, recognition site”, “cognate sequence” or “cognate sequences” refer to the minimal string of nucleotides required for a restriction enzyme to bind and cleave a DNA molecule or gene.
  • the term “enhancer region” refers to a nucleotide sequence that is not required for expression of a target gene, but will increase the level of gene expression under appropriate conditions.
  • GSH-S gene expression host selector gene
  • GSH-S refers to a genetic element that can confer to a host organism a trait that can be selected, tracked, or detected by optical sensors, PCR amplification, biochemical assays, or by cell/organism survival assays (resistance or toxicity to cells or organisms when treated with an appropriate antibiotic or chemical).
  • gene promoter or “promoter” refer to a nucleotide sequence required for expression of a gene, or any portion of the full-length promoter.
  • the terms “insert” and “module” are essentially interchangeable, with the only fine distinction being that an “insert” is inserted into the vector, and once it is inserted it is then more commonly called a “module”. A module can then be removed from the vector. Also, the term insert is commonly used for an isolated module used as an insert into a modular acceptor vector.
  • the term “intron” refers to the nucleotide sequences of a non-protein-coding region within a mammalian cell gene found between two protein-coding regions or exons.
  • LOC localization signal
  • MCS multiple cloning site
  • mRNA stabilization element refers a sequence of DNA that is recognized by binding proteins thought to protect some mRNAs from degradation.
  • ORI Olet of Replication
  • PCR terminator over-hang cloning technology refers to the process of amplifying genetic modules using the polymerase chain reaction in conjunction with single-stranded DNA primers with protected 5′over-hanging nucleotides that can serve as junction sites with complementary DNA over-hangs.
  • poly-A tail refers to a sequence of adenine (A) nucleotides commonly found at the end of messenger RNA (mRNA) molecules.
  • a Poly-A tail signal is incorporated into the 3′ ends of DNA constructs or transgenes to facilitate expression of the gene of interest.
  • primer site refers to nucleotide sequences that serve as DNA templates onto which single-stranded DNA oligonucleotides can anneal for the purpose of initiating DNA sequencing, PCR amplification, and/or RNA transcription.
  • pUC19 refers to a plasmid cloning vector well-known to those skilled in the art, and can be found in the NCBI Genbank database as Accession # L09137.
  • random nucleotide sequences refers to any combination of nucleotide sequences that do not duplicate sequences encoding other elements specified as components of the same molecule.
  • the number of nucleotides required in the random sequences is dependent upon the requirements of the endonuclease enzymes that flank the random sequences. Most endonucleases require a minimum of 2-4 additional random sequences to stabilize DNA binding. It is preferred that the number of random sequences would be a multiple of 3, corresponding to the number of nucleotides that make up a codon. The preferred minimum number of random sequences is therefore 6, however, fewer or more nucleotides may be used.
  • the term “rare” in relation to endonuclease sites refers to an endonuclease site that occurs relatively infrequently within a genome.
  • recombination arm refers to nucleotide sequences that facilitate the homologous recombination between transgenic DNA and genomic DNA. Successful recombination requires the presence of a left recombination arm (LRA) and a right recombination arm (RRA) flanking a region of transgenic DNA to be incorporated into a host genome via homologous recombination.
  • LRA left recombination arm
  • RRA right recombination arm
  • the term “recombineering” refers to the process of using random or site-selective recombinase enzymes in conjunction with DNA sequences that can be acted on by recombinase enzymes to translocate a portion of genetic material from one DNA molecule to a different DNA molecule.
  • reporter gene refers to a nucleotide sequences encoding a protein useful for monitoring the activity of a particular promoter of interest.
  • ttle Vector refers to a specialized cloning vector plasmid used in the invention to make an intermediate molecule that will modify the ends of a DNA fragment.
  • tag sequence refers to nucleotide sequences encoding a unique protein region that allows it to be detected, or in some cases, distinguished from any endogenous counterpart.
  • untranslated region refers to nucleotide sequences encompassing the non-protein-coding region of an mRNA molecule. These untranslated regions can reside at the 5′ end (5′ UTR) or the 3′ end (3′ UTR) an mRNA molecule.
  • the present invention provides a method to take a newly manufactured transgene containing the modules and selectively remove one or more of the module and replace it with a different insert. This process is called herein “second pass” and “multiple threading”.
  • the invention further provides a method for creating an array of different transgenes, each having a different Promoter, Expression and Regulatory insert, by incorporating multiple different Promoter, Expression and Regulatory inserts into a cloning vector plasmid in a single step.
  • the present invention also provides a method that comprises the steps of providing cloning vector plasmids having newly introduced Promoter, Expression and Regulatory inserts combined together, removing the entire combination as a backbone vector, and inserting a multiple number of backbone vectors into a single cloning vector plasmid.
  • the present invention also provides a method to create a modular cloning vector plasmid for the synthesis of a transgene or other complicated DNA construct by providing a backbone having docking points therein.
  • Each docking point represents an area in which there is preferably at least one fixed non-variable rare endonuclease site, and more preferably fixed groupings of two non-variable rare endonuclease sites, and most preferably fixed groupings of three non-variable rare endonuclease sites.
  • a particular restriction site of each docking point is cleaved by its cognate endonuclease enzyme.
  • At least two inserts each of which have 5′ and 3′ ends that are compatible with the cleaved docking point of interest, can be added along with the cleaved cloning vector plasmid to an appropriate reaction mixture, and, assuming the proper thermodynamic milieu, the inserts can simultaneously, i.e. in a single step, become integrated into the cloning vector plasmid.
  • the docking points are reformed and the cloning vector plasmid becomes modular, in that the docking points and the connection between the two modules can be re-cleaved with the appropriate restriction enzymes.
  • the module can then later be removed, and a new module can be put in its place.
  • One embodiment of the present invention relates to a method for constructing a transgene, comprising the steps of providing a cloning vector plasmid with a backbone able to accept a sequential arrangement of inserts, providing at least a first insert and a second insert to be included in the transgene, and transferring both the first insert and the second insert to the backbone in a single reaction. More preferably the inserts consist of three inserts, specifically at least one Promoter, Expression, and Regulatory module.
  • Another embodiment of the invention is a method for making a transgene comprising the steps of providing a cloning vector plasmid comprising first and second docking points, introducing Promoter nucleotide sequences to be included in the transgene into a Promoter shuttle vector, introducing Expression nucleotide sequences to be included in the transgene into an Expression shuttle vector, introducing Regulatory nucleotide sequences to be included in the transgene into a Regulatory shuttle vector, transferring simultaneously the Promoter, Expression and Regulatory nucleotide sequences from the Promoter, Expression and Regulatory shuttle vectors to the cloning vector plasmid, between the first and second docking points.
  • both the 5′ and 3′ ends of each of the docking points and each of the inserts all are compatible with a corresponding end of another docking point or insert.
  • a first docking point contains a restriction site for a non-variable rare restriction enzyme such as SgrAI and that docking point is thereafter cleaved
  • a first insert intended to be inserted at the 3′ end of the cleaved first docking point will contain a compatible 5′ end to create a restriction site for SgrAI when the insert is combined with the first docking point.
  • a second docking point within the plasmid may, for example, have a restriction site for a non-variable restriction enzyme such as SwaI.
  • Any second insert will have at its 3′ end a compatible nucleotide sequence to combine with the cleaved 5′ end of the cleaved second docking point to create a restriction site for SwaI. Further, the 3′ end of the first insert and the 5′ end of the second insert, in order to simultaneously be inserted into the modular cloning vector plasmid and also thereafter be removed at the same point, must contain compatible ends to create a third restriction site for a third non-variable rare restriction enzyme, such as PacI or SalI.
  • a third non-variable rare restriction enzyme such as PacI or SalI.
  • Sequential elements encoding the modular structure of the present invention can specifically comprise: three non-variable and unique common restriction sites, a 5′ oligonucleotide primer site, a unique HE site in a forward orientation, a pair of non-variable and unique, common restriction sites flanking random nucleotide sequences, a fixed grouping of non-variable rare restriction sites to define a 5′ portion of a promoter module, random nucleotide sequences, a fixed grouping of non-variable rare restriction sites that define a shared junction between a 3′ position relative to the Promoter/intron module and a 5′ position relative to an Expression module, random nucleotide sequences, a fixed grouping of non-variable rare restriction sites that define a junction of a 3′ position relative to the Expression module and a 5′ position relative to a 3′ Regulatory module, random nucleotide sequences, a fixed grouping of non-variable rare restriction sites that define a 3′ position relative to a 3′ Regulatory module
  • Other sequential elements encoding the modular structure of the present invention can specifically comprise: two non-variable and unique common restriction sites that define a 5′ insertion site, an oligonucleotide primer site, a pair of unique HE sites in opposite orientation flanking random nucleotide sequences, a non-variable and unique, common restriction site that allows cloning of a shuttle vector module downstream of the pair of unique HE sites, a fixed grouping of non-variable rare restriction sites, random nucleotide sequences, a fixed grouping of non-variable rare restriction sites, a unique HE site in a forward orientation, a pair of non-variable and unique, common restriction sites flanking random nucleotide sequences, an oligonucleotide primer site, a pair of unique BstX I sites in opposite orientations (wherein the variable nucleotide region in the BstX I recognition site is defined by nucleotides identical to the non-complementary tails generated by the ordering of two identical HE recognition
  • the present invention is a group of methods for assembling a variety of DNA fragments into a de novo DNA construct or transgene by using cloning vectors optimized to reduce the amount of manipulation frequently needed.
  • the primary vector herein referred to as a Docking Plasmid, contains a multiple cloning site (MCS) with preferably 3 sets of rare endonuclease sites arranged in a linear pattern.
  • MCS multiple cloning site
  • This arrangement defines a modular architecture that allows the user to assemble multiple inserts into a single transgene construct without disturbing the integrity of DNA elements already incorporated into the Docking Plasmid in previous cloning steps.
  • HEI-SceI cuts its cognate recognition site as indicated by “/”:
  • BstX I endonuclease enzyme site 5′CCANNNNN/NTGG 3′, where ‘N’ can be any nucleotide.
  • the sequence-neutral domain of BstX I can be used to generate compatible cohesive ends for two reverse-oriented HE protruding tails, while precluding self-annealing.
  • Endonuclease sites used in the invention were chosen according to a hierarchy of occurrence. In order to determine the frequency of endonuclease site occurrence, DNA sequence information corresponding to nineteen different genes was analyzed using Vector NTI software. This search covered a total of 110,530 nucleotides of DNA sequence. Results from these analyses were calculated according to the number of instances of an endonuclease site occurring within the analyzed 110,530 nucleotides.
  • Endonuclease sites were then assigned a hierarchical designation according to four classifications, wherein “common” sites occur greater than 25 times per 110,530 nucleotides, “lower-frequency 6 bp sites” occur between 6-24 times per 110,530 nucleotides, and “rare” sites occur between 0-5 times per 110,530 nucleotides.
  • a partial list of “suitable” enzymes is hereby listed according to their occurrence classifications:
  • Endonuclease enzymes that have a 6 bp recognition site, but have a lower frequency of occurrence:
  • the secondary vectors of the invention contain multiple cloning sites with common endonuclease sites flanked by rare endonuclease sites.
  • the shuttle vectors are designed for cloning fragments of DNA into the common endonuclease sites between the rare sites. The cloned fragments can subsequently be released by cleavage at the rare endonuclease site or sites, and incorporated into the Docking Plasmid using the same rare endonuclease site or sites found in the shuttle vectors.
  • the design of the MCS allows “cassettes” or modules of DNA fragments to be inserted into the modular regions of the Docking Plasmid. Likewise, each can be easily removed using the same rare endonuclease enzymes, and replaced with any other DNA fragment of interest. This feature allows the user to change the direction of an experimental project quickly and easily without having to rebuild the entire DNA construct.
  • the cloning vector plasmids of the present invention allow the user to clone a DNA fragment into an intermediate vector using common endonuclease sites, creating a cassette-accepting module, and to then transfer that fragment to the desired modular spot in the final construct by means of rare endonuclease sites. Furthermore, it allows future alterations to the molecule to replace individual modules in the Docking Plasmid with other cassette modules. The following descriptions highlight distinctions of the present invention compared with the prior art.
  • transgenes the promoter enhancer P, expressed protein E, and/or 3′ regulatory region 3
  • the assembled transgenes, or other nucleotide sequences can then be transferred into a Primary Docking Plasmid.
  • Each of the five types of cloning vector plasmids will be explained in greater detail to provide an understanding of the components incorporated into each, beginning with the more complex PE3 Docking Station Plasmid and the Primary Docking Plasmid.
  • the PE3 Docking Plasmid comprises a pUC19 backbone with the following modifications, wherein the sequences are numbered according to the pUC19 Genbank sequence file, Accession # L09137:
  • the Acl1 site at 1493 in pUC19 is mutated from AACGTT to AACGCT,
  • the Acl1 site at 1120 in pUC19 is mutated from AACGTT to CACGCT,
  • the Ahd1 site in pUC19 is mutated from GACNNNNNGTC to CACNNNNNGTC,
  • MCS multiple cloning site
  • a unique HE site for example, I-SceI (forward orientation)
  • RNAS-CMD-1 chromatin modification domain acceptor module
  • a fixed grouping of non-variable rare endonuclease sites that define the 5′ portion of the promoter module for example, AsiS I, Pac I, and Sbf I;
  • a fixed grouping of non-variable rare endonuclease sites that define the shared junction between the 3′ portion of the Promoter/intron module and the 5′ portion of the Expression module for example, SgrA I, AscI, and MIuI;
  • Random nucleotide sequences that can serve as a 3′ regulatory domain acceptor module (RNAS-3);
  • the Primary Docking Plasmid can be used to assemble two completed transgenes that are first constructed in PE3 Docking Station Plasmids, or two homology arms needed to construct a gene-targeting transgene, or to introduce two types of positive or negative selection elements.
  • the multiple cloning site (MCS) in the Primary Docking Plasmid comprises the following sequential elements, in the order listed:
  • RNAS-GEH-S1 A pair of unique endonuclease flanking a random nucleotide sequence of DNA that can serve as a genome expression host selector gene acceptor module (RNAS-GEH-S1);
  • a non-variable and unique, common endonuclease site that allows cloning of a shuttle vector module downstream of the HE pair (for example, EcoO109I);
  • a fixed grouping of non-variable rare endonuclease sites that define the 5′ portion a Left Recombination Arm module for example, AsiS I, Pac I, and Sbf I;
  • Random nucleotide sequences that can serve as a Left Recombination Arm acceptor module (RNAS-LRA);
  • a fixed grouping of non-variable rare endonuclease sites that define the 3′ portion of the Left Recombination Arm acceptor module for example, SgrA I, MIuI, and AscI;
  • a unique HE site for example, I-Ceu I (forward orientation)
  • RNAS-CMD-1 chromatin modification domain acceptor module
  • a pair of unique BstX I sites in opposite orientation (wherein the variable nucleotide region in the BstX I recognition site is defined by nucleotides identical to the non-complementary tails generated by the ordering of two identical HE recognition sites arranged in reverse-complement orientation; for example, PI-SceI (forward orientation) and PI-SceI (reverse orientation)) flanking a random nucleotide sequence of DNA that can serve as a complex transgene acceptor module (RNAS-PE3-1);
  • a fixed grouping of non-variable rare endonuclease sites that define the 5′ portion a Right Recombination Arm module for example, SnaB I, Sal I, and Not I;
  • Random nucleotide sequences that can serve as a Right Recombination Arm acceptor module (RNAS-RRA);
  • a non-variable and unique, common endonuclease site that allows cloning of a shuttle vector module (for example, BspE I);
  • RNAS-GEH-S2 genome expression host selector gene acceptor module
  • Three cloning vector plasmids of the invention are known as Shuttle Vectors.
  • the Shuttle Vectors like the PE3 and Primary Docking Plasmids, are also constructed from a pUC19 backbone. Just like the PE3 and Primary Docking Plasmids, each Shuttle Vector has the same modifications to the pUC19 backbone listed as 1 through 6, above.
  • the individual Shuttle Vectors (SV) are identified as Shuttle Vector Promoter/intron (P), Shuttle Vector Expression (E), and Shuttle Vector 3′Regulatory (3); henceforth SVP, SVE, and SV3, respectively. Each is described more fully below.
  • SVP is a cloning vector plasmid that can be used to prepare promoter and intron sequences for assembly into a transgene construct.
  • An example of an SVP Plasmid can comprise the following sequential elements in the MCS, in the order listed:
  • a non-variable and unique, common endonuclease site that allows efficient cloning of a shuttle vector module downstream of the T7 primer site (for example, Eco0109I);
  • a fixed grouping of non-variable rare endonuclease sites that define the 5′ portion of the promoter module for example, AsiSI, Pac I, and Sbf I. These non-variable rare endonuclease sites provide the docking point represented by the star at the 5′ end of the Promoter Vector of FIG. 2 ;
  • variable MCS comprising any grouping of common or rare endonuclease sites that are unique to the shuttle vector
  • a fixed grouping of non-variable rare endonuclease sites that define the 3′ portion of the promoter module for example, SgrA I, AscI, and MiuI. These non-variable rare endonuclease sites provide the docking point represented by the circle at the 3′ end of the Promoter Vector of FIG. 2 ;
  • a non-variable and unique, common endonuclease site that allows efficient cloning of a shuttle vector module upstream of the T3 primer site (for example, BspEI);
  • An example of an SVE plasmid can comprise the following sequential elements in the MCS, in the order listed:
  • a non-variable and unique, common endonuclease site that allows efficient cloning of a shuttle vector module downstream of the T7 primer site (for example, Eco0109I);
  • a fixed grouping of non-variable rare endonuclease sites that define the 5′ portion of the expression module for example, SgrA I, AscI, and MIuI. These non-variable rare endonuclease sites provide the docking point represented by the circle at the 5′ end of the Expression Vector of FIG. 2 ;
  • variable MCS consisting of any grouping of common or rare endonuclease sites that are unique to the shuttle vector
  • non-variable rare endonuclease sites that define the 3′ portion of the expression module (for example, SnaBI, NotI, and SalI). These non-variable rare endonuclease sites provide the docking point represented by the triangle at the 3′ end of the Expression Vector of FIG. 2 ;
  • a non-variable and unique, common endonuclease site that allows efficient cloning of a shuttle vector module upstream of the T3 primer site (for example, BspEI);
  • An example of an SV3 plasmid can comprise the following elements in the MCS, in the order listed:
  • a non-variable and unique, common endonuclease site that allows efficient cloning of a shuttle vector module downstream of the T7 primer (for example, Eco0109I);
  • a fixed grouping of non-variable rare endonuclease sites that define the 5′ portion of the 3′ regulatory module for example, SnaBI, NotI, and SalI. These non-variable rare endonuclease sites provide the docking point represented by the triangle at the 5′ end of the Regulatory Vector of FIG. 2 .
  • variable MCS consisting of any grouping of common or rare endonuclease sites that are unique to the shuttle vector
  • a fixed grouping of non-variable rare endonuclease sites that define the 3′ portion of the 3′ regulatory module for example, SwaI, RsrII, and BsiWI. These non-variable rare endonuclease sites provide the docking point represented by the square at the 3′ end of the Regulatory Vector of FIG. 2 ;
  • a non-variable and unique, non-rare endonuclease site that allows efficient cloning of a shuttle vector module upstream of the T3 primer site (for example, BspEI);
  • transgenes in plasmid cloning vectors similar methods can be used to build transgenes in larger extrachromosomal DNA molecules such as cosmids or artificial chromosomes, including bacterial artificial chromosomes (BAC).
  • BAC bacterial artificial chromosomes
  • T1 vector may also be used.
  • the wide variety of genetic elements that can be incorporated into the plasmid cloning vectors also allow transfer of the final transgene products into a wide variety of host organisms with little or no further manipulation.
  • FIGS. 2 and 3 are a general illustration of the modularity of the invention. As shown in FIG. 2 , there is one each of a Promoter, Expression, and 3′ Regulatory shuttle vector. Flanking each insert within the shuttle vectors are endonuclease restriction sites that are specific for creating a docking point. More specifically, in FIG.
  • the Promoter insert (P) is flanked by a first group of one or more endonuclease restriction sites represented by astar at the 5′ end and a second group of one or more endonuclease restriction sites represented by a circle at the 3′ end;
  • the Expression module is flanked by the second group of endonuclease restriction sites represented by the circle at the 5′ end and a third group of one or more endonuclease restriction sites represented by a triangleat the 3′ end;
  • the 3′ Regulatory module (3) is flanked by the third group of endonuclease restriction sites represented by the triangle at the 5′ end and a fourth group of one or more endonuclease restriction sites represented by a square at the 3′ end.
  • each endonuclease recognition site by the endonuclease specific for that site creates sticky ends at the 5′ and 3′ end of each module, as indicated in bottom portion of FIG. 2 by the inserts at the end of the dashed line arrows.
  • the modules can now be combined with a cloning vector plasmid which has also been cleaved at its two fixed docking points by endonucleases specific for the first group of endonuclease restriction sites (represented by the star) and the fourth group of endonuclease restriction sites (represented by the square).
  • the cleaved sticky ends represented by hollow stars, circles, triangles and squares
  • the cleaved star ends will self-orient within the plasmid and sequentially ligate, with the cleaved star ends combining, the cleaved circle ends combining, the cleaved triangle ends combining, and the cleaved square ends combining. This results in an assembled backbone vector shown in FIG. 3 .
  • each of the combined groups of endonuclease sites represented by the star, circle, triangle, and square can once again be cleaved by its corresponding specific endonuclease, such that a particular insert can later be removed and replaced with another insert of interest.
  • backbone vectors can be inserted into a single docking plasmid.
  • the sequence-neutral domain of BstX I can be used to generate compatible cohesive ends for two reverse-oriented I-Sce I protruding tails, while precluding self-annealing.
  • a first insert, PE3-1, having an I-Sce-1 site at its ends can be placed in a cloning vector plasmid by cleaving the plasmid at the Bstx1/Sce1 endonuclease sites.
  • One can then cut again with I-SceI and insert a PE3-2 having I-Sce-1 site at its ends.
  • This entire backbone can then be cleaved from its docking plasmid by PI-Sce I and inserted into another docking plasmid that contains BstX I/PI-Sce I endonuclease sites.
  • This second docking plasmid also has endonuclease sites for PI-Sce I, into which yet another module for a separate docking plasmid, possible containing a PE3-3 and PE3-4, can be inserted (not shown).
  • a researcher can get more information into one cell, that is, one can insert multiple genes within the context of a single vector, which has not previously been accomplished by those skilled in the art.
  • Such a novel process can save a both money and time for researchers working in this field.
  • transgene can be constructed containing these elements:
  • the SP-C sequences contain internal BamH1 sites, and can be released from its parental plasmid only with Not1 and EcoR1.
  • GMR ⁇ c has an internal Not1 site, and can be cut from its parental plasmid with BamH1 and Xho1.
  • the rabbit betaglobin intron sequences can be cut out of its parental plasmid with EcoR1.
  • the SV-40 poly-A tail can be cut from its parental plasmid with Xho1 and Sac1. Because of redundancy of several of endonuclease sites, none of the parental plasmids can be used to assemble all the needed fragments.
  • the steps used to build the desired transgene in the PE3 Docking Plasmid invention are as follows.
  • rabbit betaglobin intron sequences are cloned into the EcoR1 site of pSVP-SPC. Orientation of the intron in the resultant intermediate construct is verified by sequencing the product, called pSVP-SPC-r ⁇ G.
  • the promoter and intron are excised and isolated as one contiguous fragment from pSVP-SPC-r ⁇ G using AsiS1 and Asc1. Concurrently, the PE3 Docking Plasmid is cut with AsiS1 and Asc1 in preparation for ligation with the promoter/intron segment. The promoter/intron fragment is ligated into the Docking Plasmid, propagated, and recovered.
  • the Xho1 site of the GMR ⁇ c fragment is filled in to create a blunt 3′ end, using techniques well known to those skilled in the art. It is then cloned into the BamH1 site and the blunt-ended Pvu2 site of pSVP-SPC-r ⁇ G. The resultant plasmid (pDP-SPC-GMR ⁇ -r ⁇ G) was propagated and recovered.
  • the final cloning step is the addition of the SV-40 Poly-A tail.
  • the SV40-polyA fragment is cut out with Xho1 and Sac1, as is the recipient vector pDS1-SPC-GMR ⁇ c-rb ⁇ G. Both pieces of DNA are gel purified and recovered.
  • a ligation mix is prepared with a 10:1 molar ratio of SV-40polyA to pDS1-SPC-GMR ⁇ c-r ⁇ G. The ligation products are propagated and harvested.
  • the new plasmid, pDS1-SPC-GMR ⁇ c-r ⁇ G-pA contains all elements required for the transgene, including a unique endonuclease site at the 3′ end with which the entire pDS1-SPC-GMR ⁇ c-r ⁇ G-pA plasmid can be linearized for transfection into eukaryotic cells or microinjection into the pronucleus of a fertilized ovum.
  • Promoter sequences from the human cytomegalovirus (CMV) are inserted into a P Shuttle Vector (SVP), having AsiSI and Ase I endonuclease at the 5′ and 3′ portions, respectively. Plasmids are amplified, and the promoter module is cleaved from the vector by AsiS I and Asc I endonuclease digestion and isolated.
  • SVP P Shuttle Vector
  • Sequences encoding a luciferase protein are inserted into an Expression Shuttle Vector (SVE), having Asc I and Not I endonuclease at the 5′ and 3′ portions, respectively. Plasmids are amplified, and the Expression module is cleaved from the vector by Asc I and Not I endonuclease digestion and isolated.
  • SVE Expression Shuttle Vector
  • Sequences encoding a mammalian intron and SV40 poly-adenylation site are inserted into a 3′ Regulatory Shuttle vector (SV3), having Not I and BsiW I endonuclease at the 5′ and 3′ portions, respectively. Plasmids are amplified, and the Regulatory module is cleaved from the vector by Not I and BsiW I endonuclease digestion and isolated.
  • SV3 3′ Regulatory Shuttle vector
  • the Promoter, Expression, and Regulatory modules are combined with the Docking Vector Plasmid in a ligation mixture. Following an incubation of 2 hours, the ligation mixture is used to transform E. coli , which are then spread on an LB agar plate with ampicillin. The plate is incubated at 37° C. overnight. Colonies are isolated and propagated in individual liquid LB broth cultures. The plasmid DNA is isolated from each LB broth culture. The DNA is analyzed by endonuclease mapping to determine whether the plasmids from each colony contain the three modular inserts (Promoter, Expression and Regulatory). A plasmid that contains the three modular inserts is identified as the transgene pCMV-luc-SV40 pA.
  • CMV-luciferase mice can be linearized using I-Sce I endonuclease and injected into mouse pronuclei to generate CMV-luciferase mice.
  • the CMV promoter in this example directs the expression of the luciferase gene in all tissues of a host organism, such as a CMV-luciferase mouse.
  • a neuron-specific promoter Neuron-Specific Enolase (NSE)
  • NSE Neuron-Specific Enolase
  • SVP P Shuttle Vector
  • pCMV-luc-pA is cleaved with AsiS I and Asc I to remove the CMV Promoter Module. The remainder of the Docking Vector Plasmid containing intact Expression and Regulatory Modules is isolated.
  • the NSE Promoter Module is placed in a ligation mixture with the remainder of the Docking Vector Plasmid containing intact Expression and Regulatory Modules. Following incubation for 2 hours, the new ligation mixture is used to transform E. coli .
  • the E. coli mixture is spread on an LB agar plate with ampicillin, as in the previous example. Colonies are isolated the following day, propagated, and plasmid DNA is isolated from each. Endonuclease mapping is used to identify plasmids that contain the desired NSE Promoter module.
  • the following example is an illustration of the use of the invention to rapidly assemble an array of transgenes, each containing a different combination of Promoter, Expression, and Regulatory modules.
  • a series of six shuttle vectors and a PE3 docking station vector will be used to generate eight different vector products using combinatorial assembly.
  • the series of six shuttle vectors consists of two P-Shuttles (SVP), two E-Shuttles (SVE), and two 3-Shuttles (SV3).
  • the two discrete P-Shuttles (SVP) contain either a human cytomegalovirus (CMV) promoter or a mouse SPC lung-specific promoter, and each has AsiS I and Asc I endonuclease at the 5′ and 3′ portions, respectively.
  • CMV human cytomegalovirus
  • Asc I endonuclease at the 5′ and 3′ portions, respectively.
  • the two discrete E-Shuttles contain either a Luciferase cDNA or an EGFP cDNA, and each has Asc I and Not I endonuclease at the 5′ and 3′ portions, respectively.
  • the two discrete 3-Shuttle vectors contain either an SV40 polyA signal or the 3′ regulatory region of the human growth hormone (hGH), and each has Not I and BsiW I endonuclease at the 5′ and 3′ portions, respectively.
  • the promoter modules are released from their respective SVP shuttle vectors by individually digesting appropriate shuttle vector with the AsiS I and the Asc I endonucleases.
  • the resulting restriction products are individually subjected to gel electrophoresis and the DNA band corresponding to the appropriate promoter module is subjected to gel purification. This procedure will yield either a CMV promoter module or an SPC promoter module bounded on the 5′ side by an AsiS I overhang and by an Asc I overhang on the 3′ end.
  • the expression modules are released from their respective SVE shuttle vectors by individually digesting appropriate shuttle vector with the Asc I and the Not I restriction endonucleases.
  • the resulting restriction products are individually subjected to gel electrophoresis and the DNA band corresponding to the appropriate expression module is subjected to gel purification.
  • This procedure will yield either a Luciferase expression module or an EGFP expression module bounded on the 5′ side by an Asc I overhang and by a Not I overhang on the 3′ end.
  • the 3′ regulatory modules are released from their respective SV3 shuttle vectors by individually digesting appropriate shuttle vector with the Not I and the BsiW I restriction endonucleases.
  • the resulting restriction products are individually subjected to gel electrophoresis and the DNA band corresponding to the appropriate 3′ regulatory module is subjected to gel purification.
  • This procedure will yield either a SV40 3′ regulatory module or an hGH 3′ regulatory module bounded on the 5′ side by a Not I overhang and by a BsiW I overhang on the 3′ end.
  • the PE3 docking station vector is prepared by digesting with the AsiS I and the BsiW I restriction endonucleases. To help prevent future vector re-ligation, the vector restriction digest is exposed to calf intestinal phosphatase (CIP) for one hour at 37° C. The resulting CIP-treated vector restriction product is then subjected to gel electrophoresis and the DNA band corresponding to linearized PE3 vector backbone is subjected to gel purification.
  • CIP calf intestinal phosphatase
  • Samples from the seven resulting gel-purified DNA fragments are analyzed for identity, integrity, purity, and quantity by running out on a diagnostic electrophoretic gel. Quantitative data concerning the relative abundance of the purified PE3 docking station vector and the respective DNA modules is used to define the amount of each component needed for a combinatorial ligation reaction.
  • the first strategy is to set up ligation reaction mixtures wherein the insert-to-vector ratio is about 3:1.
  • the second strategy, used when more than one insert is being introduced to a single vector simultaneously, is to introduce a molar equivalent of each genetic module that will be inserted into the vector. This can be achieved either by adding a variable volume of the modules to a reaction container in order to obtain molar equivalence in the context of the ligation reaction mixture, or by adding a neutral buffer solution to each of the purified modules so that their concentrations are equivalent on a molar ratio basis.
  • the gel-purified vector and insert fragments have all been adjusted to molar equivalence using the buffer 10 mM Tris, pH 8.0.
  • the total ligation reaction volume is set at 150 microliters.
  • the ligation reaction mixture consists of the following constituents: 39 microliters of ultrapure water, 15 microliters of 10 ⁇ Ligase buffer, 5 microliters of the purified PE3 vector backbone, 15 microliters of the purified CMV Promoter module, 15 microliters of the purified SPC Promoter module, 15 microliters of the purified Luciferase expression module, 15 microliters of the purified EGFP expression module, 15 microliters of the purified SV40 3′ regulatory module, 15 microliters of the purified hGH 3′ regulatory module, and 1 microliter of ligase enzyme.
  • the resulting reaction components are thoroughly mixed and then incubated overnight at 16° C.
  • the predicted vector ligation products include the following:
  • the ligation mixture is then used to transform E. coli , which are then spread on an LB agar plate with ampicillin. The plate is incubated at 37° C. overnight. Colonies are isolated and propagated in individual liquid LB broth cultures. The plasmid DNA is isolated from each LB broth culture. The DNA is analyzed by endonuclease mapping to determine the identity of the resulting vector incorporated into each colony.
  • one of the predicted vector products was not produced during the first combinatorial process.
  • One vector that was successfully produced can, however, serve as a vector backbone for producing the desired pCMV-EGFP-SV40 vector. This technique can be referred to as “Second Pass Assembly”.
  • the pCMV-Luciferase-SV40 vector product of Example 4 is digested with Asc I and Not I, CIP-treated, and subsequently gel-purified.
  • This linearized vector fragment, in which the Luciferase module has been removed, is incubated in a ligation mixture containing the EGFP module produced in the previous example of combinatorial vector assembly.
  • the ligation mixture is used to transform E. coli , which are then spread on an LB agar plate with ampicillin. The plate is incubated at 37° C. overnight. Colonies are isolated and propagated in individual liquid LB broth cultures. The plasmid DNA is isolated from each LB broth culture. The DNA is analyzed by endonuclease mapping to determine whether the plasmids from each colony contain the EGFP insert.
  • both Dynamic Vector Assembly in which one each of a Promoter, Expression and Regulatory insert can be inserted into a single backbone at the same time, and the combination method described, in which two P-Shuttles, two E-Shuttles, and two Regulatory-Shuttles are all combined to create eight different types of transgenes, can be used to save precious time and money for researchers.
  • Shuttles that were originally created by de novo synthesis, recombineering, and PCR terminator over-hang cloning methods can be taken and used with the docking point technology of the present invention to rapidly assemble these pre-made elements into a multitude of transgenes.
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EP2484772B1 (de) 2016-08-17

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