Classifications

• • 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
• • 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
  • C12N2800/00—Nucleic acids vectors
  • C12N2800/20—Pseudochromosomes, minichrosomosomes
  • C12N2800/208—Pseudochromosomes, minichrosomosomes of mammalian origin, e.g. minichromosome

Definitions

• the present invention relates to methods for introducing (multiple) transgenes into a primary cell as well as to methods for producing (multi-)transgenic primary cells, tissues and/or animals by utilizing nucleic acid constructs that are capable of de novo formation of artificial chromosomes.
• the present invention furthermore relates to uses of these (multi-)transgenic primary cells, tissues and animals, preferably in the context of xenotransplantation.
• the nucleic acid constructs capable of de novo forming artificial chromosomes are utilized as versatile and effective means for the transfer of (multiple) human transgenes and, thus, ultimately for the generation of animals as organ donors in xenotransplanation.
• Xenotransplantation is the transplantation of living cells, tissues or organs from one species to another, such as from pigs to humans. Such cells, tissues or organs are called xenografts or xenotransplants.
• Human xenotransplantation offers a potential treatment for end-stage organ failure, a significant worldwide health problem, in particular in parts of the industrialized world. Because there is a worldwide shortage of organs for clinical implantation, about 60% of patients awaiting replacement organs die on the waiting list. However, xenotransplantation also raises many novel medical, legal and ethical issues. Immune rejection remains the biggest challenge for xenotransplantation. The problem exists even for human to human transplants, but is more serious for transplants between different species.
• Chromosome-based vector systems offer two primary advantages over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression.
• telomeres for protection and maintenance of chromosome ends
• replication origins for DNA duplication
• centromere for segregation at cell division
• centromere is a complex structure with multiple roles in the control of segregation at cell division, including assembly of kinetochore and spindle attachment, maintenance of sister chromatid cohesion until anaphase onset and movement of chromosomes to opposite poles.
• alpha satellite DNA consists of a approx. 171 bp repeating unit that can exist either as tandemly arranged monomers (monomeric alpha satellite DNA) or as multimeric groups that have been amplified to form chromosome-specific higher order repeats (of typically several megabases). Higher order alpha satellite forms the major part of the alpha satellite array at all human centromeres.
• telomeric DNA to sequentially truncate human chromosomes and generate smaller derivative minichromosomes
• bottom up the generation of de novo human artificial chromosomes from naked DNA transfected into cultured human cells
• Harrington et al, 1997 disclose the first-generation system for the construction of human artificial chromosomes (HAC), wherein long synthetic arrays of alpha satellite DNA were combined with telomeric DNA and genomic DNA to generate artificial chromosomes in human HTl 080 cells, a telomerase positive, human lung sarcoma cell line showing a pseudostable, largely pseudotetraploid karyotype.
• HAC human artificial chromosomes
• the resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in the absence of selection for up to six months in culture, bind centromere proteins specific for active centromeres, and are estimated to be 6-10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes.
• YAC yeast artificial chromosome
• the 100-kb YAC-based system contained human centromeric DNA (of the human chromosome 21 centromere region of suprachromosoaml family II) as well as telomere repeats and selectable markers.
• This YAC which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E.
• the MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism.
• Mammalian artificial chromosomes (MACs) as well as HACs represent a versatile, non- integrating gene delivery system with a sufficient cloning capacity for the inclusion of multiple gene loci.
• the de novo construction of MACs is based on the transfection of large constructs of "naked" DNA leading to the formation of stably segregating, low copy episomes.
• One of the most important components is a functional centromere. It was found that the process of de novo centromere formation on HACs in cultured HTl 080 tumor cells only worked efficiently with members of the primary, evolutionary modern class of human alpha satellite DNA, which is composed of long homogeneous tandem repeat arrays and contains binding sites for the centromere binding protein B (CENP-B).
• CENP-B centromere binding protein B
• centromere DNA of chromosome 5 representing the first example for a member of human suprachromosomal family I
• all suprachromosomal families complying with these criteria (evolutionarily modern, homogeneous tandem repeat array, and containing CENP-B boxes) formed centromeres efficiently.
• the present invention aims to improve the methods and means for the transfer of multiple genes into primary cells and for the generation of trangenic animals as present in the prior art and it is, thus, an objective of the present invention to provide improved gene transfer means and methods utilizing these gene transfer means in order to allow an efficient and safe transfer of multiple genes into primary cells and to allow the generation of multi-transgenic animals, which are especially suitable in xenotransplantation and can also be used to screen non-Mendelian genetic diseases and develop multi-transgenic cell and gene therapy.
• this object is solved by providing a method for introducing transgenes into a primary cell, preferably a method for introducing multiple transgenes into a primary cell.
• the method for introducing (multiple) transgenes into a primary cell according to the present invention preferably comprises the following steps
• transgene at least one transgene, preferably at least two transgenes
• this object is furthermore solved by providing a (multi)- transgenic primary cell obtained by the method for introducing (multiple) transgenes into a primary cell according to the invention.
• this object is furthermore solved by providing a de novo formed artificial chromosome obtained by the method for introducing (multiple) transgenes into a primary cell according to the invention.
• this object is furthermore solved by providing the use of the (multi-)transgenic primary cell and/or the de novo formed artificial chromosome for producing (multi-)transgenic animals, preferably mammals, producing (multi-)transgenic cells, tissues and organs for (xeno)transplantation.
• this object is furthermore solved by providing a method for producing (multi-)transgenic animals or birds, preferably mammals (except humans).
• the method for producing (multi-)transgenic animals or birds according to the present invention preferably comprises the following steps
• transgene at least one transgene, preferably at least two transgenes
• this object is furthermore solved by providing a (multi)- transgenic animal or bird and/or a (multi-)transgenic tissue obtained by the method for producing (multi-)transgenic animals or birds according to the present invention.
• this object is furthermore solved by providing a use of the (multi-)transgenic animal or bird: as donor for the xenotransplantation of organs, tissues, cells, cell mass, neo-organs as screening model for gene therapy, drug screening for the production of proteins and metabolites for the production of vaccines for grafts detoxifying metabolites or antibodies for the production of biomass and biomaterials for the investigation of non-Mendelian diseases for determining the copy number of specific genes and the function of the copy numbers in the development of said non-Mendelian diseases for gene therapy in form of providing a respective copy number of said specific genes
• the (multi-)transgenic animal or bird is preferably a (multi-)transgenic mammal, more preferably a (multi-)transgenic pig.
• the present invention provides a method for introducing transgenes into a primary cell, preferably a method for introducing multiple transgenes into a primary cell.
• multi-transgenic refers to more than or at least two, preferably more than or at least 3 transgenes that are contained within a nucleic acid construct and/or introduced into a cell or into cells of a tissue, organ, animal.
• the present invention also comprises nucleic acid constructs that comprise at least one transgene, preferably multiple copies of one transgene.
• a nucleic acid construct which is capable of de novo forming an artificial chromosome.
• a "de novo formed artificial chromosome” according to the invention is an independent, non- integrating, stably segregating genetic unit which replicates as a low copy element, preferably with 1 to 2 copies, and which during cell division is exactly divided and segregates exactly to the daughter cells.
• the de novo formed artificial chromosomes are preferably stably inherited for preferably at least 100 cell divisions in the absence of selection.
• the de novo formed artificial chromosome preferably assembles inside the transfected cells and the deriving cell line and segregates efficiently alongside the host genome
• nucleic acid construct "capable of de novo forming an artificial chromosome” is a nucleic acid construct which after introduction into a cell, such as by transfection or lipofection, will lead to the de novo formation of an independent, non-integrating genetic unit in form of an artificial chromosome.
• nucleic acid constructs of the invention utilize the "bottom up” approach for generating artificial chromosomes and not the “top down” or minichromosome approach, i.e. they are not sequentially truncated human chromosomes.
• the de novo formation of artificial chromosomes in a host cell can lead to or result in an addition of sequences, such as larger stretches of telomeres, from the cell into the de novo formed artificial chromosome due to recombination and/or repair processes.
• the de novo formation of artificial chromosomes refers to the self assembly of an inheritable chromatin structure that can stably segregate in the host cell, based on a nucleic acid construct and numerous chromatin components present within the host cell (proteins, histones, nuclear factors), and as such is defined as, and aims at the formation of the novel, surplus genetic unit solely from the input nucleic acid molecule/s and sequences present in the transferred nucleic acid constructs.
• repair/recombination leading to a replicating structure is at work in the cell.
• This process principally can lead to an uptake of sequence portions of the host, for example by recombination, more likely involving reverse transcription, such as healing and lengthening of the provided telomere sequences by longer host telomere copies in telomerase negative cell types.
• Such non-integrative (in terms of stable integration into a host chromosome) repair processes might also occur at non-telomeric sequence portions, and therefore presence of such (repetitive, or repeat bound) additional sequence portions (if at all) does not affect the "de novo" state.
• de novo formed structures containing such additional sequences are also within the scope of this invention.
• a “nucleic acid” according to the invention is DNA, RNA, nucleic acids with modified nucleosides.
• the "nucleic acid" according to the invention is DNA.
• a nucleic acid construct of the invention contains long, defined, purified molecules of undamaged DNA.
• Such a DNA construct can be further modified, such as by methylation, or can contain modified nucleosides.
• a nucleic acid construct of the invention can comprise DNA and RNA.
• DNA can be supplemented with RNA or DNA or supportive DNA constructs (transient) transcribed to an RNA other than the construct itself, or an RNA or a DNA producing an RNA sequence of the long DNA construct, or the long DNA construct plus additional RNA copies of the long (genomic) DNA construct/and or portions thereof (which serves as a repair/replication substrate during chromatinization), improving clone formation, or any kind of supportive RNA encoding growth factors and proteins to increase clone formation and/or improve initial chromatinization of the HAC.
• the nucleic acid construct is of very high quality, more preferably of a Grade 1 DNA preparation.
• a “Grade 1 DNA preparation” refers to a preparation of DNA molecules of preferably long DNA, wherein “long” refers to DNA of longer than about 20 kb, wherein the majority of molecules of the preparation is intact DNA, more preferably more than 90% of the preparation is intact DNA, preferably more than 95%, more preferably more than 99%, up to 100% intact, i.e. DNA free of breaks.
• a "Grade 1 DNA preparation” refers to a preparation of a store of DNA molecules, which in its entirety (vast majority) of long (> 20 kb) DNA molecules is free of breaks and nicks (double strand breaks (dsb) and single strand breaks (ssb)).
• the fact that the DNA molecules are stored and later obtained with the high quality allows the use of functionally defined DNA constructs.
• the preferred Grade 1 DNA quality increases physical stability of long molecules for subsequent handling. This allows a reduction of the load of free DNA ends per functioning and complete molecule, as well as improves initial enzymatic read- through by repair and replication polymerases within the primary cell and/or stem cell.
• the nucleic acid constructs of the invention are preferably produced by utilizing a method for the in vitro production of long nucleic acid constructs, which is called in gel site specific recombination (IGSSR). This method is described in detail in US 6,331,397 as well as DE 197 20 839 B4 (of the inventor) and in Schindelhauer and Cooke, 1997, which are incorporated herein by reference in their entirety.
• IGSSR gel site specific recombination
• the IGGSR method for producing a nucleic acid construct comprises the steps of providing a recombinase; providing nucleic acids (DNAs) for recombination, each nucleic acid (DNA) comprising a sequence specific for the recombinase; providing an agarose at a temperature which maintains the agarose in a sufficiently liquid state to permit sufficiently homogeneous mixing of the nucleic acids (DNAs) and the recombinase to permit recombination of the nucleic acids (DNAs) to form the nucleic acid (DNA) construct; combining the recombinase and the nucleic acids (DNAs) in the agarose for a time and under conditions sufficient to effect recombination of the nucleic acids (DNAs) to form the nucleic acid (DNA) construct; and separating the nucleic acid (DNA) construct from the other nucleic acid (DNA) in the agarose.
• the methods preferably results in solidified agarose gel comprising
• nucleic acids with other, non-genomic transgenes are also suitable for the present invention.
• This also refers to any transfections using conventional DNA preparations (such as alkaline lysis, conventional plasmid preparations, mixtures of preparations), crude extracts, or DNA that has not been stored and analysed, constructs inside transfer bacteria or DNA in cells used for direct transfer, or transfer by fusion of cells with a less well defined cellular nucleic acid content, but which harbour the nucleic acid constructs according to this invention.
• conventional DNA preparations such as alkaline lysis, conventional plasmid preparations, mixtures of preparations
• crude extracts or DNA that has not been stored and analysed
• constructs inside transfer bacteria or DNA in cells used for direct transfer, or transfer by fusion of cells with a less well defined cellular nucleic acid content but which harbour the nucleic acid constructs according to this invention.
• the nucleic acid construct of the invention comprises as a single unit (i) sequence(s) conferring telomere functions, (ii) centromeric region,
• origin element(s) conferring unit copy replication (iv) at least one gene selectable in prokaryotes , (v) at least one gene selectable in eukaryotes, (vi) at least one transgene, preferably at least two transgenes, (vii) restriction site(s), (viii) optional further features.
• a "sequence conferring telomere function" refers to a nucleotide sequence which acts like a chromosomal telomere, i.e. a nucleotide sequence which serves to protect and maintain the chromosome ends by reverse transcription and, thus, protects the nucleic acid construct after introduction into cells (e.g. by transfection) from integration.
• the sequences conferring telomere function further serve to hide free DNA ends, allowing progress through the cell cycle after transfer of the DNA molecule without integration or degradation of the construct.
• sequence(s) conferring telomere function (i) of the nucleic construct of the invention are preferably selected from telomeric repeat(s), telomeric region(s) and telomere-like structure(s).
• sequence(s) conferring telomere functions are at least one per nucleic acid construct, but can also be more, such as 2, 3, 4, 5 and more sequence(s) conferring telomere functions.
• sequence(s) conferring telomere functions are two telomeric sequences in opposite direction, i.e. they are arranged in opposite orientation on the nucleic acid construct (e.g. referring to the G-rich sequence of the nucleotide sequence, e.g. TTAGGG) and/or preferably additional subtelomeric telomere repeats.
• sequence(s) conferring telomere function are repeats forming
• sequence(s) conferring telomere function comprise 3 or 4 or up to 10 telomere sequences of tandem repeats, which can be interspersed.
• Each repeat or tandem repeat contains repetitive consensus units of (TTAGGG)n, which is the only known telomeric tandem repeat consensus sequence presently known in all vertebrates and also present in some lower eukaryotes and plants.
• (TTAGGG)n is also comprised, i.e. (CCCTAA)n.
• tandem repeat of several telomeric consensus sequences/repetitive consensus units is then referred to as a "repeat array” or “tandem repeat array", wherein the repetitive consensus units (e.g. TTAGGG) are in the same orientation in the array.
• the sequences conferring telomere functions of the nucleic acid construct of the invention comprise preferably 2, 3, 4, 5 and more of such repeat arrays or tandem repeat arrays.
• the complementary sequence of TTAGGG i.e. CCCTAA is also comprised.
• the nucleic acid construct of the invention comprises two sequences conferring telomere functions in opposite direction, preferably each comprising 3 or more repetitive consensus units of (TTAGGG)n (wherein n is 3 or more, such as 135).
• the complementary sequence of TTAGGG is also comprised.
• telomere reversely transcribed repeat sequences for example arrays of repetitive sequence elements like retroposons serving telomere replication and maintenance, as known of drosophila TA elements, or protein capped linear DNA structures, hairpin ends, circular structures or circular HAC configurations are used to confer or substitute telomere function.
• arrays of repetitive sequence elements like retroposons serving telomere replication and maintenance, as known of drosophila TA elements, or protein capped linear DNA structures, hairpin ends, circular structures or circular HAC configurations are used to confer or substitute telomere function.
• the centromeric region (ii) of the nucleic construct of the invention is preferably selected from a centromeric region of a human chromosome. Such a centromeric region is capable of de novo forming a functional centromere.
• the centromeric region of the nucleic construct of the invention is preferably selected from human centromeric alpha-satellite DNA, more preferably selected from human alpha satellite DNA belonging to one of the suprachromosomal families I, II, or III (preferably with an evolutionarily modern, homogeneous tandem repeat array), preferably of a satellite sequence found to efficiently form de novo centromeres.
• animal satellite sequences showing the species-specific characteristics of an evolutionarily modern, homogeneous satellite array of a centromere on a normal chromosome in this species can be used as a centromere sequence in the invention (i.e. as centromeric region (U)).
• the centromeric region (ii) is preferably a centromeric region of human chromosome 5 belonging to suprachromosomal family I, more preferably a section containing approximately 341 repeat units of the about 340 bp alpha satellite dimers of chromosome 5, more preferably comprising an array of about 116 kb (El) or an array of other size, containing repeats with identical, or containing repeats with a high similarity, to the dimeric repeats sequenced herein, which are preferably derived from construct TTEl directly as Eco RI subcloned repeats (see SEQ ID NOs. 23, 24) or of the construct TTEl that have been propagated stably in pigs and isolated thereafter by PCR for sequencing (see SEQ ID NOs. 15-22).
• the centromeric region (ii) is preferably a centromeric region of human chromosome 5 of suprachromosomal family I, preferably a section containing approximately 341 repeat units of the about 340 bp alpha satellite dimers of chromosome 5, more preferably comprising an array of about 116 kb (El), even more preferably comprising a sequence selected from SEQ ID NOs. 15 to 24.
• the nucleic construct comprises one centromeric region (ii), but can also comprise more of such centromeric regions, e.g. 2, 3, 4, 5 and more.
• the centromeric region(s) are for replication and exact segregation at cell division.
• centromeric sequences like telomeric sequences if contained in multiple copies, might also serve other chromosomal functions, like as chromatin boundaries, insulators, or elements supporting replication in the absence of genomic replication origins, which usually are not identified on a sequence level.
• An "origin element conferring unit copy replication” refers to any nucleic acid sequence contained within a nucleic acid construct which serves the regulated replication once per cell division.
• An "origin element conferring unit copy replication” is a nucleic acid sequence working within it's host cell to allow one replication initiation per cell generation, leading to a stable low copy number, usually one per host chromosome (unit copy number).
• Single or unit copy replication allows to determine the function of a single nucleic acid construct molecule per cell (because cells do not show a potential mixture of many functioning and not functioning molecules, as it can be the case with high copy plasmids, which would lead to difficulties in sequencing and functional characterizations on a single molecule level).
• the low abundance of the nucleic acid construct molecules with a unit copy replicon reduces unwanted inter-molecule recombinations, which could take place between the chromosomal repetitive sequence elements and the common intergenic and intragenic repeats within genomic transgenes.
• unit copy replication improves nucleic acid construct stability and sequence conformity within the DNA preparation of a clone (and, thus, improves construct quality).
• the origin element(s) conferring unit copy replication (iii) of the nucleic construct of the invention is preferably a unit copy replicon, such as unit copy replicon of phage Pl or F factor.
• the origin element(s) conferring unit copy replication (iii) of the nucleic construct of the invention within a prokaryotic/bacterial host is preferably a unit copy replicon, such as unit copy replicon of phage Pl or F factor.
• unit copy replicon such as unit copy replicon of phage Pl or F factor.
• replicons provide in addition to using the normal host factors for chromosome replication and segregation, genes encoding an ATPase and a DNA binding protein and a DNA sequence to bind to, serving as a replication start and a prokaryotic centromere at once, both together allowing a regulated number of replications of the artificial chromosome per replicated host genome (unit copy), and an exact segregation of the replicated chromosomes to both daughter cells.
• the Pl replicon is preferred and is suitable for stable cloning of long genomic genes and centromers into the nucleic acid construct.
• the nucleic acid constructs of the invention furthermore comprise sequences or genes that allow a selection in prokaryotic and/or eukaryotic cells and hosts, i.e. gene(s) selectable in prokaryotes and/or eukaryotes. These sequences or genes are referred to as "gene selectable in prokaryotes", “gene selectable in eukaryotes” or "selection genes”.
• the nucleic acid construct of the invention comprises at least one gene selectable in prokaryotes (iv), which is preferably located (resides) in between the sequences conferring telomere functions in an embodiment where more than one sequence conferring telomere function is present in the nucleic acid construct of the invention.
• the nucleic acid construct of the invention comprises two (or more) genes selectable in prokaryotes, wherein one gene is preferably located in between the sequences conferring telomere function and the other gene is preferably located outside the sequences conferring telomere functions (i.e. within the vector backbone).
• additional selectable genes (3, 4, 5 and more) can be placed in between each of the telomeric regions (independent of the orientation of the flanking telomeric regions), increasing cloning stability of each intertelomeric segment.
• a multi-telomeric construct contains multiple (different) selectable genes supporting cloning stability.
• Preferred genes selectable in prokaryotes are usually selected from antibiotic resistance genes.
• Genes selectable in prokaryotes are known in the art. The skilled artisan will be able to select respective gene(s) selectable in prokaryotes depending on a specific application of the present invention.
• genes selectable in prokaryotes are:
• the nucleic construct comprises one or two gene(s) selectable in prokaryotes, but can also comprise more of such genes, e.g. 3, 4, 5 and more.
• the at least one gene selectable in eukaryotes (v) of the nucleic construct of the invention is preferably selected from resistance genes, fluorescence genes, but is not limited to such
• selection genes can include genes allowing a phenotypical selection or selection by affinity.
• Genes selectable in eukaryotes are known in the art. The skilled artisan will be able to select respective gene(s) selectable in eukaryotes depending on a specific application of the present invention.
• genes selectable in eukaryotes are:
• BS Blasticidin S
• the nucleic construct comprises one or two gene(s) selectable in eukaryotes, but can also comprise more of such genes, e.g. 3, 4, 5 and more.
• the transgene(s) (vi) of the nucleic acid construct of the invention are preferably at least one transgene, more preferably at least two transgenes, more preferably at least 3 transgenes, but can also be 4, 5 and more transgenes.
• the transgene(s) are furthermore preferably at least one genomic transgene locus.
• the transgenes are selected from eukaryotic genes in natural or altered form, preferably human genes and factors or genes/factors adapted to the respective human genes/factors.
• a (trans)gene in an "altered form” refers to an artificial and/or deliberately engineered alteration or modification of the gene/protein product and/or the nucleotide sequence not commonly found as a naturally occuring variant. Fragments of the (trans)gene or defect forms of the (trans)gene (which do not lead to a gene/protein product) are comprised.
• a (trans)gene in "natural form” or “unchanged form” means that a nucleic acid sequence is used reflecting a normal gene locus (section of a normal chromosome) and that the transgene locus section used expresses a naturally occuring protein variant, which does not necessarily need to be the common form and can also be a less functioning, a more functioning variant, or a rare variant found in nature with other or additional special functional features.
• the transgenes are in a naturally occurring form and contain all exons and all introns.
• the transgenes are used in the form of DNA sections including upstream and downstream regulatory regions and regulatory regions within and in between the exons as a genomic gene locus section, i.e. gene loci that can replicate structurally stably and efficiently form expressing chromatin.
• the nucleic acid constructs of the invention comprise: multiple unchanged transgenes, multiple transgenes including altered genes, multiple genes to generate multi-transgenic model animals, including animals with multiple copies of disease genes.
• Transgenes in other forms such as a gene solely based on a cDNA expression cassette, also fall within the scope of the present invention.
• the transgenes are selected from human genes, animal genes, eukaryotic genes.
• the transgenes are not confined to the typical members/genes suitable for xenotransplantation, described below and in Table 2, and can also be artificial, or viral or prokaryotic, or of a parasite, or modified of a parasite escaping the immune system effectively and carrying factors/proteins which induce tolerance such as the intracellular protozoa leishmania, or compound engineered genes, for example to escape the immune system.
• one of the multiple transgenes is a defect (altered) gene fragment of porcine origin, which can be advantageous for a reduced immunogenic surface or specific binding of immunosuppressants or blocking antibodies.
• the transgenes are selected from transgenes suitable for (xeno)transplantation or from subsets for (xeno)transplantation, preferably from human genes or genes adapted to humans suitable for xenotransplantation or from subsets for xenotransplantation, such as
• complement regulators such as membrane bound DAF (CD55), MCP (CD46), CRl (CD35), CD 59, or indirectly, variants of soluble proteins involved in autoimmune conditions like factor H, C4b binding protein,
• immunomodulators such as CTLA4-Ig, co-stimulating factors, INDOl, co-stimulation blockers, TNFalpha related apoptosis inducing ligand TRAIL, co-stimulating factors, programmed cell death ligands, PD-Ll
• blood clotting modulators such as human thrombomodulin hTM, NTPDase CD 39, hTFPI, human heme oxygenase hHO-1,
• inhibitors of mRNA such as siRNAs and their expression cassettes
• surface modulating enzymes such as human glycosyltransferases (sialyl-, fucosyl-)
• health increasing proteins such as proteins protecting from pre-existing disease states of an organ/organ system, including such as of Mendelian genetic diseases (cystic fibrosis, myopathies, storage diseases) and non-Mendelian genetic diseases (diabetes, kidney failure, heart failure, angiopathy, immune mediated diseases, blood pressure).
• Mendelian genetic diseases cystic fibrosis, myopathies, storage diseases
• non-Mendelian genetic diseases diabetes, kidney failure, heart failure, angiopathy, immune mediated diseases, blood pressure.
• the transgenes are selected from genes producing therapeutic proteins, enzymes and factors, such as blood clotting factors, and proteins defect in Mendelian genetic diseases like hemophilia including hemophilia A and B, factor XIII, factor IX deficiency, myopathies, dystrophies, cystic fibrosis, storage diseases, lysosomal storage diseases (approximately 40), including those caused by deficiencies of soluble lysosomal enzymes, including GAA deficiency (Morbus Pompe), a deficiency frequently involved in muscle weakness of the elderly, which can be cured by expression and excretion of supraphysiologic enzyme levels in transplantable cells, tissues and organs, for adding multiple surplus therapeutic genes and for the excretion of supportive metabolites, and in primary cells for gene therapy.
• therapeutic proteins like hemophilia including hemophilia A and B, factor XIII, factor IX deficiency, myopathies, dystrophies, cystic fibrosis, storage diseases, lysosomal storage diseases (approx
• the transgenes are selected from genes producing multiple therapeutic proteins including the production of therapeutic proteins and biomasses.
• transgenes or subsets of transgenes on a multigenic unit such as the nucleic construct of the invention
• the surfaces of the cells (and, thus, tissues and organs) of a transgenic animal will be protected from being rejected, in particular after (xeno)transplantation of said cells, tissues and organs.
• transplanted organ/tissue/cell mass restores health by suppressing the immune response, or expressing systemic factors and proteins or metabolites lacking in the recipient, for example regulated insulin levels of pancreatic islets.
• transplanted organ/tissue/cell mass is used to not be rejected transiently but expresses proteins for vaccination and induction of an immune response, or to alleviate autoimmune disease by attraction of the immune system.
• transgenes Based on an average genomic gene size of 21-12 kb, up to 10-20 transgenes can technically be realized in a primary cell using the present invention and technology based on a pTAT, pTT, pTTEl vector, grade 1 DNA, and in gel site specific recombination for the directed joining of long DNA molecules beyond anticipated cloning limits of 0.3-0.5 Mb in such vectors.
• the nucleic construct comprises at least two transgenes, more preferably at least 3 transgenes, but can also comprise 4, 5 and more transgenes.
• the number of transgenes will depend on the application of the present invention.
• the nucleic acid construct can also comprise more than one copy of the same transgene and/or marker gene.
• restriction site(s) (vii) are preferably rare cutter restriction site(s), restriction site(s) for cloning inside the sequences conferring telomere functions and/or restriction site(s) for cloning of transgenes, and optionally, white blue selectable restriction site(s) for the cloning of genomic libraries, and/or sequence(s) for in vitro site-specific recombination.
• Restriction sites are known to the skilled person in the art. The skilled artisan will be able to select respective restriction site(s) according to a specific application of the present invention.
• restriction sites are:
• Bsp 120 I and Bss H II e.g as cloning sites between the telomeres for inserting of centromere genes or DNA tags
• l-Sce I sites 1, 2, 3, 4 (or more) l-Sce I sites and other intron encoded rare cutter restriction sites, which are not or extremely rarely cutting within eukaryotic genomes (I-Sce I introduced at the tips of the telomeres to release telomerized fragments, one large, or more fragments depending on the number of telomeres).
• At least one of several possible rare cutter restriction sites which frequently cut the E. coli genome and which are very rare in genomic DNA regions of animals, mammals, humans, and therefore are usually absent in the transgenes, are preferably absent in the entire nucleic acid construct of the invention.
• Asc I, Rsr II are absent in the pTAT vectors, in order to allow efficient production of Grade 1 DNA.
• rare cutters predicted to not cut in vector pTT include Fse I, SgrA I, Srfl.
• Presence of duplicated vector DNA portions in vector pTT, and pTTEl between the telomeres e.g. in order to facilitate homologous repair within a single transferred molecule.
• the size of the nucleic acid construct is preferably more than 100 kb, preferably more than 120 kb.
• the size of the nucleic acid construct is preferably more than 100 kb, preferably more than 120 kb and also depends on the number and size of the transgenes.
• the size of the nucleic acid construct is preferably more than 133 kb in form of the linear, telomerized and centromere 5 containing construct pTTEl, depending on the number and size of additional transgenes. A rough estimate would be a size increase of 50 kb per additional transgene.
• the size of the nucleic acid construct is principally without any known size limit. According to present practicability, up to a size of perhaps 0.6-1 Mb, however, the skilled artisan will be able to achieve larger sizes once improved methods will be available.
• the nucleic acid construct capable of de novo forming an artificial chromosome is preferably a vector or plasmid, more preferably a HAC vector construct.
• the vector or plasmid is preferably a BAC or PAC, more preferably a HAC vector construct.
• telomeric DNA to sequentially truncate human chromosomes and generate smaller derivative minichromosomes
• bottom up the generation of de novo human artificial chromosomes from naked DNA transfected into cultured human cells
• a preferred HAC vector construct is derived from pTAT, pTT, pTTEl, and derivatives preferably containing one site-specific recombination sequence, such as loxP, of at least one type, but can contain more types of non-interfering specific recombination sequences (FRT, modified).
• the vector or plasmid has been isolated from a large circular DNA preparation, preferably BAC or PAC (or less preferred a circular YAC), within a gel matrix, preferably low melting agarose, that has been purified from broken and nicked molecules > 20 kb and is free of breaks in the vast majority of molecules of a preparation (Grade 1 quality).
• a large circular DNA preparation preferably BAC or PAC (or less preferred a circular YAC)
• a gel matrix preferably low melting agarose
• nucleic acid construct is introduced into a primary cell.
• a "primary cell” according to the present invention is selected from primary cells of an animal, preferably a mammal, more preferably a pig, or a bird.
• animals also comprise birds.
• a "primary cell” according to the present invention is derived from animals or animal breeds which are preferred or particularly suitable for xenotransplantation, due to their organ size, pre-set natural or engineered tissue/cell parameters, and safety considerations.
• animals which have been domesticated for thousands of years pig, sheep, goat
• many humans have lived in close contact including numerous direct tissue and cell contacts (by nutrition or injuries)
• Organ size limitations and compatibility of physiologic parameters might also limit the choice of an organ for xenotransplantation, for example pig hearts from smaller pig breeds could fit better to humans.
• a "primary cell” according to the present invention is preferably derived/isolated from the following animals or animal breeds:
• the primary cell is selected from bone marrow cells, fibroblasts, adipocytes, endothelial cells, microvascular endothelial cells, progenitors of the cells, stem cells, including germ line, and cells of an organ, tissue, cell mass, cell type to be transplanted.
• the source of the primary cells can be live animals (intraoperative, or small sample), slaughtered animals, cell samples, tissue samples, organs (including blood, germ line, reproductive organs, sperm fluid), primary cell cultures, ektodermal, endodermal, mesodermal, including stem cells, embryo, fetus, newborn animal, young animal, adult animal.
• the introduction is preferably by transfection, lipofection or by in situ methods followed by isolation of transfected primary cells and cultivation using lipoplexes, polyplexes, magnetofection, transfer via bactofection, or sperm mediated transfer.
• lipoplexes polyplexes
• magnetofection transfer via bactofection
• sperm mediated transfer a method for introducing large nucleic acid constructs into cells and will be able to determine the respective introduction method after studying the present disclosure.
• a (multi-)transgenic primary cell is obtained.
• step (d) of the method of the invention the de novo formed artificial chromosome is obtained.
• the artificial chromosome obtained can be used - to study structure and function, thus further identifying quality and functionality of the nucleic acid construct used, preferably of a stored batch of grade 1 DNA of the construct used for subsequent further construction/modification of the nucleic acid construct.
• the functional artificial chromosome can be moved/transferred to other cells, either isolated from cells, or in the form of cell fusion, or fusion of micronuclei containing the artificial chromsome, or other methods known to the skilled artisan.
• the method for introducing (multiple) transgenes into a primary cell preferably comprises the following steps
• sequence(s) conferring telomere functions selected from telomeric repeat(s), telomeric region(s) and telomere-like structures, preferably two sequences conferring telomere functions in opposite direction, preferably each comprising repetitive consensus units of (TTAGGG)n, wherein n is 3 or more, (ii) centromeric region, preferably of a human chromosome, (iii) origin element(s) conferring unit copy replication preferably a unit copy replicon, such as unit copy replicon of phage Pl or F factor,
• transgene at least one transgene, and preferably at least one genomic transgene locus, preferably at least two transgenes, more preferably at least 3 transgenes,
• the present invention provides a (multi-)transgenic primary cell obtained by the above method according to the invention.
• the (multi-)transgenic primary cell is preferably obtained from an expanding cell clone as the result of a low copy molecule (preferably 1 or 2) transfer event (in situ or in culture) derived from a single cell, preferably selectable during expansion for the presence of a stable genetic unit (using selection in culture, or later selection of derived animals presenting stable HAC).
• a low copy molecule preferably 1 or 2 transfer event (in situ or in culture) derived from a single cell, preferably selectable during expansion for the presence of a stable genetic unit (using selection in culture, or later selection of derived animals presenting stable HAC).
• the (multi-)transgenic primary cell comprises preferably a de novo formed artificial chromosome.
• the present invention provides a de novo formed artificial chromosome obtained by the above method according to the invention or from a (multi-)transgenic primary cell obtained by the above method according to the invention.
• the present invention provides a method for producing transgenic animals or birds, preferably a method for producing multi-transgenic animals or birds.
• animals also comprise birds.
• the method produces preferably a (multi-)transgenic mammal, preferably selected from domesticated animals, such as pig, sheep, cow, water buffalo, goat, horse, donkey, reindeer, dog, cat, ferret, mink, rabbit, hare, camel, llama, alpaca, dromedary, mouse, rat, chinchilla, hamster, guinea pig, and other mammals, or chicken, goose, duck, turkey, ostrich and other birds.
• domesticated animals such as pig, sheep, cow, water buffalo, goat, horse, donkey, reindeer, dog, cat, ferret, mink, rabbit, hare, camel, llama, alpaca, dromedary, mouse, rat, chinchilla, hamster, guinea pig, and other mammals, or chicken, goose, duck, turkey, ostrich and other birds.
• the mammal is preferably a pig.
• Each species or breed is providing an individual potential for the modelling of genetic diseases (Mendelian and common non-Mendelian) according to inherited individual disease susceptibilities, and can show an individual physiologic and size restricted compatibility of organs, safety parameters, and risk parameters for transplant rejection.
• the method is not for providing (multi-)transgenic humans.
• the method produces a (multi-)transgenic pig.
• a nucleic acid construct is provided which is capable of de novo forming an artificial chromosome.
• nucleic acid construct and preferred embodiments thereof are described herein above and below.
• nucleic acid construct is introduced into a primary cell of an animal.
• the primary cell of an animal is selected from bone marrow cells, fibroblasts, adipocytes, endothelial cells, microvascular endothelial cells, progenitors of the cells, stem cells, including germ line, and cells of an organ, tissue, cell mass, cell type to be transplanted.
• the introduction is preferably by transfection into cultured primary cells, or by in situ methods followed by isolation of primary cells and cultivation using lipoplexes, polyplexes, magnetofection, transfer via bactofection, or sperm mediated transfer.
• lipoplexes polyplexes
• magnetofection transfer via bactofection, or sperm mediated transfer.
• the skilled artisan is familiar with methods and procedures for introducing large nucleic acid constructs into primary cells of an animal and will be able to determine respective suitable introduction methods after studying the present disclosure.
• a (multi-)transgenic primary cell is obtained.
• the (multi-)transgenic primary cell is preferably obtained from an expanding cell clone as the result of a low copy molecule (preferably 1 or 2) transfer event (in situ or in culture) derived from a single cell, preferably selectable during expansion for the presence of a stable genetic unit (using selection in culture, or later selection of derived animals presenting stable HAC).
• a low copy molecule preferably 1 or 2 transfer event (in situ or in culture) derived from a single cell, preferably selectable during expansion for the presence of a stable genetic unit (using selection in culture, or later selection of derived animals presenting stable HAC).
• offspring of said animal is produced by using said (multi-)transgenic primary cell.
• the offspring is preferably produced by nuclear transfer using nuclei of the transfected primary cell clones (with or without selection), which are introduced into enucleated oocytes and implanted into female recipients resulting in a pregnancy and derived cloned mutitransgenic offspring.
• the method for producing (multi-)transgenic animals according to the present invention preferably comprises the following steps
• nucleic acid construct capable of de novo forming an artificial chromosome comprising as a single unit, as defined herein, (b) introducing said nucleic acid construct into a primary cell of an animal, preferably a mammal,
• the present invention provides a (multi-)transgenic animal obtained by the method for producing (multi-)transgenic animals according to the present invention.
• the (multi-)transgenic animal is preferably a mammal (except a human), more preferably a Pig-
• the (multi-)transgenic animal preferably comprises a de novo formed artificial chromosome, which is formed from the nucleic acid construct described herein, in each cell of the animal. More preferably, all of the multiple transgenes provided by the nucleic acid construct described herein are contained and preferably expressed in each cell of the animal, according to the regulated chromatin context specific to a given cell type.
• the present invention also provides a (multi-)transgenic tissue obtained from the multi-transgenic animal of the invention or from the (multi-)transgenic primary cell of the invention.
• the (multi-)transgenic tissue preferably comprises a de novo formed artificial chromosome.
• the present invention provides uses of the de novo formed artificial chromosome, the (multi-)transgenic primary cell, the (multi-)transgenic tissue and/or (multi)- transgenic animal obtained by the methods of the invention.
• nucleic constructs of the invention which are (multigenic) units comprising any desired transgenes or subsets of transgenes (in particular transgenes that are suitable in the context of xenotransplantation), can be used to modify the surfaces of the cells (and, thus, tissues and organs) of a transgenic animal in order to be protect from rejection, in particular after (xeno)transplantation of said cells, tissues and organs.
• multi-transgenic cells for individualized medicine is an important aspect of this invention.
• Xenotransplantation and transplantation of artificial organs, cells, tissues, neo- organs from a defined animal source, which are fitted with mutliple transgenes for individual disease states, offers a manageable alternative to obtaining primary cells of patients and subsequent expansion in cell culture.
• the production of biomaterials and therapeutic cell devices from an animal source provides the possibility to explore, calculate, and define a risk, and can easily generate very large numbers of cells, tissues and organs.
• the animal source has an advantage over cells of a patient, which need to be expanded in cell culture for many cell divisions and each time pose a new risk of developing cancer and/or of rejection.
• the generation of a larger number of patient cells, apart from the technical burden, is time and cost intensive.
• a safe generation and unlimited growth of stem cells of each patient is not available.
• the use as a source of adapted muti-transgenic animal cells with a defined risk to generate transplantable cell devices and organs for multiple purposes represents a workable technology in terms of versatility, costs, and approval.
• the (multi-)transgenic primary cell and/or the de novo formed artificial chromosome obtained by the methods of the invention are preferably used for producing (multi-)transgenic animals, preferably mammals producing (multi-)transgenic tissues, transplantation of cells, tissues and/or organs.
• the (multi-)transgenic primary cell and/or the de novo formed artificial chromosome obtained by the methods of the invention are preferably used for producing (multi-)transgenic animals, preferably mammals producing (multi-)transgenic tissues, transplantation of cells, tissues and/or organs, producing biomass, generating transplantable biomaterials, generating organs, including encapsulated or artificial organs, neo-organs for the excretion of metabolites, factors, proteins or to detoxify metabolites or inactivate antibodies, expressing vaccines.
• the (multi-)transgenic animal preferably mammal, more preferably pig, obtained by the methods of the invention is preferably used:
• the (multi-)transgenic animal, preferably mammal, more preferably pig, obtained by the methods of the invention is preferably used for the isolation of o therapeutic organs, o therapeutic tissues, o therapeutic cells, o therapeutic proteins, - the production of o therapeutic cells, o multi-transgenic therapeutic organs, o proteins in transplantable cells, o therapeutic proteins, o metabolites in transplantable cells o vaccines o biomaterial or biomass from multi-transgenic animals, o multi-transgenic herds, o therapeutic organs (exchange and additional), - the study of disease and/or protection genes and generating therapeutic cells.
• biomass/biomaterial refers to material obtained from the multi-transgenic animal that is depleted or free of cells or contains only inactivated cells.
• the biomass/biomaterial includes e.g. bone matrix, wound-healing paste or glue, fascia, suture material, coating and the like, and preferably contains suitable proteins, is not rejected, and can be re-colonized with animal or human cells.
• Metal excreting factories refers to living, transplantable supportive devices, including organs, partial organs, encapsulated or artificially structured organs to produce proteins, metabolites, vaccines, or to detoxify metabolites, or attract antibodies/autoimmune disease, and is health improving or protecting.
• non-therapeutic uses are within the scope of the invention, such as multi-transgenic livestock, like for enhancement performance of animals and pet animals (such as look of pet animals, fur colour, competitive racing).
• An enhancement of livestock animals and other animals comprises the improvement of health of the animals using the multi-transgenic artificial chromosomes of the invention, which include genes and gene variants identified at QTLs (Quantitative Trait Loci), including identification/verification of QTL candidate genes.
• HACs large cloning capacity allows the introduction of a wide range of gene loci without the size limitations associated with the currently used, integrating vectors (see e.g. Grimes et al. 2001, Mejia et al. 2001).
• a way of generating artificial chromosomes is their de novo formation upon transfection of isolated chromosomal elements (Harrington et al. 1997, Ikeno et al. 1998).
• the de novo approach aims at the development of pre-fabricated HAC vectors, which contain all sequence elements required for replication, segregation, and expression.
• the construct can be used to further join additional sequence elements and desired gene loci, which is particularly useful for the multiple genes required for xenotransplantation. Therefore, HAC development also included the improvement of molecular tools for the precise construction of long DNA assemblies from suitable genomic resources, such as BACs and PACs.
• BACs and PACs Two principally different strategies to build up the desired sequences have been devised.
• Several groups rely on a quick joining of the functional sequence elements and gene loci using a modification of PACs/BACs inside living E. coli cells based on random or targeted recombination (see e.g. Mejia et al. 2000).
• the inventor developed a technology to build up long DNA constructs using conventional cloning in PACs.
• the long DNA technology includes the isolation of intact DNA (>100 kb) and subsequent manipulation, such as biochemical joining (Schindelhauer and Cooke 1997; US 6,331,397; DE 197 20 839), or very long PCR (presently up to 64.7 kb of human genomic DNA, DE 10 2004 025 650 of the inventor) to introduce restriction sites via primers, and for improving functional transfer of long DNA (Laner et al. 2005).
• a prerequisite of MAC construction is the functional assessment of all sequence elements to be included. Expression on HACs has been demonstrated via complementation of a genetic deficiency in the host cell line used (Grimes et al. 2001, Mejia et al. 2001, Ikeno et al. 2002).
• SusAC pig (sus) artificial chromosome
• HAC vector construct pTTEl an embodiment of an nucleic acid construct
• the pTT construct has the following features:
• TTAGGG telomere sequences
• the vector construct pTTEl used in a Embodiment and the Example disclosed herein has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on March 31, 2008 under Accession number DSM 21324.
• telomerised PACs Pl artificial chromosome for the propagation of long genomic DNA in E. coli
• PACs Pl artificial chromosome for the propagation of long genomic DNA in E. coli
• the human genes and factors and genes and factors adapted to humans shall protect the cell surface and organs against rejection. Suitable therefor are, for example, complement factors (CD46 and others), coagulation modulators (thrombomodulin and others, CD39), co-stimulating factors (PD-Ll) and other toleration factors, immunomodulators CTLA, cytokines ILlO, IL23, chemokines, HLA-E, HLA class I, II, III antigens, modified antigens, si-RNAs, surface-modulating enzymes, and -in a broader sense- inhibitors of endogenous viruses (PERV) and modified immunogenic proteins, as well as health-increasing protein variants against a pre-existing cell/organ disease.
• complement factors CD46 and others
• coagulation modulators thrombomodulin and others, CD39
• co-stimulating factors PD-Ll
• a human artificial chromosome with a centromere of chromosome 5 was used in a pig in order to determine whether the telomerised construct would also lead to a stably inheritable unit in pig primary cells and would allow the development of a whole animal organism.
• said human artificial chromosome it was known that it can efficiently form a centromere and lead to an autonomous artificial chromosome in human tumor cells (Laner et al 2004, 2005), wherein artificial chromosomes had already successfully introduced 3 gene portions into human tumour cells stably and at the same time (genomic HPRT or CFTR gene, EGFP marker gene, BS resistance gene).
• the intensity corresponded substantially with the intensity of the original cell clone.
• the green fluorescence was maintained during the further expansion, which practically corresponds to a 100% stability.
• a re-selection with Blasticidin S with a part of the cell cultures showed an almost 100% survival rate of the fibroblast cultures of both pigs, only single cells of the about 10 5 re-selected cells detached, whereas practically all cells resumed growing after an initial recovering phase of a few days and stably fluoresced green.
• DAPI stained metaphase chromosomes of the fibroblast culture were counted and a normal chromosome set was counted. Again and again, small surplus DAPI elements could be seen located closely to the normal chromosomes or free.
• genomic PCR of the vector and specifically for chromosome 5 satellite sequences were performed (Primer 5IFopt/5IR, for details see Examples). Both sequences were present in the genomic DNAs, but could not be detected in the DNA from non-transfected cells.
• FISH fluorescence in situ hybridization
• a method can be used based on these and following artificial chromosomes.
• custom tailored units can now be produced in primary cells, which can be passed functionally without selection. Since the construction takes place on DNA level, the major development of xenotransplantation can be performed independently of certain pig strains.
• HACs human artificial chromosomes
• telomerized PAC construct TTEl (142 kb) bearing 116 kb of the dimeric alpha satellite DNA family "El" of human chromosome 5 was known to efficiently form artificial chromosomes in HTl 080 cells (Laner et al, 2004; Laner et al, 2005).
• telomerized PAC vector pTT (26 kb) contains (one of the two) blasticidin S (BS) and the CMV-EGFP expression cassette on a 6 kb sub/inter-telomeric fragment not containing a prokaryotic selection marker (Laner et al, 2004; Laner et al, 2005), and therefore can become partially lost during bacterial growth, not impairing HAC formation but reducing the rate of green clones.
• the DNA batch of TTEl used in the present invention contained the EGFP marker in roughly 2/3 of the physically intact molecules (grade 1 DNA), as has been estimated from transfections with construct mixtures with and without EGFP (data not shown).
• bone marrow cells were collected from the large bones and ribs of a 5 months old boar (80 kg) from the slaughterhouse.
• the cells were purified from blood on a Ficoll gradient (Pretlow et al, 1968) and expanded in T75 tissue culture flasks for 9 days in DMEM containing 10% FCS, Amphotericin B, Penicillin, and Streptomycin.
• DMEM fetal calf serum
• Fibroblasts of both pigs were cultured for 36 d off selection or 7 d off and 29 d on BS re- selection ( Figures 8A-D), which did not result in an increased cell death or reduced growth, as judged under the microscope.
• Those and later passages after expansion from frozen stocks were subjected to fluorescence in situ hybridization (FISH).
• FISH fluorescence in situ hybridization
• HAC detection was based on presence of both signals, the human centromere 5 probe "El” (pink) and either EGFP (red) or BS vector probe "rsf ' (Laner et al, 2005) (red), and absence of integration of both probes into a pig chromosome, which was shown for both pigs in all probe combinations on and off selection (Figure 5C).
• porcine centromeric MCl (Roger-Gaillard et al, 1997) satellite derived probe "smf/r" (for further information see Example) which strongly hybridized to several metacentrics and weakly to some of the acrocentrics (green), excluded presence of most (2/3) of the pig centromere regions on the HACs.
• An attempt to isolate an acrocentric probe based on published repeat sequences did not result in a specific satellite PCR pattern or useful probe, and therefore could not be excluded on the HACs.
• Attempts to amplify a potential human-pig satellite junction with primer combinations of smf/r with 5IF/R and 5IFopt were negative in both pigs after 35 cycles and 4 min elongation (data not shown).
• CD39 is the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase). It converts ATP and ADP to AMP, which is further degraded to the antithrombotic and antiinflammatory mediator adenosine
• Figure 1 Schematic description of the fate of transgenic DNA after transfection into eukarytic cells.
• Telomere, centromere, and transgene sequences are pre-fabricated to long DNA molecules and transferred to primary cells, in order to de novo form stably segregating, non-integrating artificial chromosomes.
• the capability of the transfected cell to subsequently enter the cell cycle, divide and expand to a clone expressing mutiple components, (critically) depends on a high quality of long DNA molecules, containing all functional components within each molecule, and not in broken portions, as regularly only very few (usually one, sometimes two, but rarely more than two transferred molecules can participate in faithful chromatinization and clone formation (even if using transfer methods physically transferring larger numbers of molecules to the cells initially, like lipofection).
• DNA preparations with a large fraction of unbroken molecules are preferred in this invention.
• the vector also contains prokaryotic resistance genes kana (in the vector backbone, not shown) and amp (in the HAC construct, not shown), unique restriction sites including white blue selectable Sal I site (in the HAC construct, not shown) for the cloning of genomic libraries and lox P sequences (not shown).
• prokaryotic resistance genes kana in the vector backbone, not shown
• amp in the HAC construct, not shown
• unique restriction sites including white blue selectable Sal I site (in the HAC construct, not shown) for the cloning of genomic libraries and lox P sequences (not shown).
• FIG. 3 Stable primary porcine cell clones obtained by lipofection of HAC construct. Shown are six different single cell transfer events surviving BS selection and expanding to clonal lines from lipofections of construct pTTEl into primary sus scrofa mesenchymal, ssMC (A) and sus scrofa microvascular endothelial cells, ssMVEC. Left columns: light microscopic view; right columns: fluorescence microscopic view. Images were captured on a Zeiss Axiovert 25 at varying magnification, equipped with a HBOlOOW lamp and a colour CCD camera, and processed on Axiovision software.
• Figure 4 First animals obtained from primary bone marrow cell clone transfected with a multi-transgenic HAC construct.
• the green fluorescent and well growing primary cell clone seg4 derived by transfection of HAC construct TTEl has been subjected to nuclear transfer and resulted in two healthy green piglets 9562 and 9563.
• Top row pig 9562
• middle and lower row pig 9563.
• Amphotericin B Penicillin, Streptomycin. Please note that green fluorescence was observed in virtually all cells under the microscope. Weakly expressing cells in the vicinity of bright sections appear dark in some sections shown, not necessarily indicating a lack of EGFP expression.
• HAC de novo formed HAC is an independent, non-integrating genetic unit.
• a Metaphases derived from skin fibroblasts grown for 7d off and 39 d on BS showed the normal number of 38 chromosomes, as counted in 7 out of 8 complete looking metaphase spreads of pig 9563, out of which 5 showed a free little DAPI element (arrowhead), reflecting a normal detection rate and being compatible with full stability.
• B Karyogram of pig 9563 (signals are from a FISH analysis to discriminate pig centromere probes not relevant here).
• Figure 9 PCi? analysis of genomic samples of the primary cell clones and skin cells derived from the transgenic piglets.
• a EGFP PCR was used to check genomic DNA samples and stable presence of the vector sequences as expected from the strong green fluorescence under the microscope.
• human alpha satellite repeats usually present at > 1000 copies per human genome was amplified in a 34 cycle PCR using primers X-3A/4A. No products were obtained from the pig DNA samples, which were adjusted to contain equal amounts of approximately 100 ng of genomic DNA per reaction (EthBr staining).
• Figure 10 Schematic overview for a preferred method of producing long nucleic acid constructs in agarose gel blocks.
• the data demonstrate a meiotic transmission rate close to normal inheritance of one chromosome (of two paired chromosomes) and suggest feasibility of generating larger herds with human artificial chromosomes.
• Example 1 Artificial chromosomes formed in primary pig cells and in piglets derived from these primary cells.
• bone marrow cells were collected from the large bones and ribs of a 5 months old boar (80 kg) from the slaughterhouse.
• the cells were purified from blood on a Ficoll gradient (Pretlow et al, 1968) and expanded in T75 tissue culture flasks for 9 days in DMEM containing 10% FCS, Amphotericin B, Penicillin, and Streptomycin.
• DMEM fetal calf serum
• Fibroblasts of both pigs were cultured for 36 d off selection or 7 d off and 29 d on BS re- selection ( Figures 8A-D), which did not result in an increased cell death or reduced growth, as judged under the microscope.
• Those and later passages after expansion from frozen stocks were subjected to fluorescence in situ hybridization (FISH).
• FISH fluorescence in situ hybridization
• HAC detection was based on presence of both signals, the human centromere 5 probe "El” (pink) and either EGFP (red) or BS vector probe "rsf ' (Laner et al, 2005) (red), and absence of integration of both probes into a pig chromosome, which was shown for both pigs in all probe combinations on and off selection (Figure 5C).
• porcine centromeric MCl (Roger-Gaillard et al, 1997) satellite derived probe "smf/r" (for further information see Example) which strongly hybridized to several metacentrics and weakly to some of the acrocentrics (green), excluded presence of most (2/3) of the pig centromere regions on the HACs.
• An attempt to isolate an acrocentric probe based on published repeat sequences did not result in a specific satellite PCR pattern or useful probe, and therefore could not be excluded on the HACs.
• Attempts to amplify a potential human-pig satellite junction with primer combinations of smf/r with 5IF/R and 5IFopt were negative in both pigs after 35 cycles and 4 min elongation (data not shown).
• FIG. 4 shows a light photography at day 7 and under a blue lamp (9V, 20 cm spot) at day 1.
• Figure 5B top row: pig 9562, middle and lower row: pig 9563).
• the single surplus DAPI element observed in all analyses showed both, a signal for vector probe rsf or EGFP (red) and the human centromere 5 alpha satellite input DNA El (pink).
• HAC (arrowhead, merged channels) and single channel sections with the HAC at the right
• EGFP expression in skin tissue sections of both piglets obtained at day 7 after birth and various cell types grown out for 7 d in DMEM, FCS, Amphotericin B, Penicillin, Streptomycin are shown in Figure 6. Please note that green fluorescence was observed in virtually all cells under the microscope. Weakly expressing cells in the vicinity of bright sections appear dark in some sections shown, not necessarily indicating a lack of EGFP expression.
• Figure 8 shows EGFP expression in cultured fibroblasts from skin samples after 36 d of growth without selection or 7 d off and 29 d on BS. Brightness of EGFP expression was somewhat increased in the selected cells, however the vast majority of cells grown off selection were faint green as observed by eye under a Zeiss 10 microscope equipped with a HBO50 lamp.
• human alpha satellite repeats usually present at > 1000 copies per human genome was amplified in a 34 cycle PCR using primers X-3A/4A. No products were obtained from the pig DNA samples, which were adjusted to contain equal amounts of approximately 100 ng of genomic DNA per reaction (EthBr staining) (see Figure 9C).
• the primers have been used to generate the pig centromeric satellite probe smf/r.
• Primer 5IFopt was derived from sequenced samples of dimers of construct TTEl and contains three different nucleotide positions compared to primer 5IF (which was used to isolate a PAC clone containing the El array insert that has been subcloned in pTTEl). In combination with primer 5IR both lead to products of 0.275 kb / 0.275 + 0.34 kb, typical for the D5Z2 alpha satellite array present on human chromosome 5. Primers 5IF/R were published in Laner et al. 2005.
• EGF/R primers amplify a 281 bp EGFP coding portion and were published in Laner et al. 2005.
• X-3A/4A primers amplify a X chromosome specific 0.5 kb alpha satellite product from human genomic DNA. Locus DXZl usually bears > 1000 highly similar copies per chromosome. Primers were published in Warburton and Willard 1992. - Sequences used and generated in this invention
• Centromere sequences of the El type have been subcloned from TTEl (AM409269, AM409268, Eco RI subclones, see SEQ ID NOs. 23 and 24) and 5IF/R PCR products obtained from genomic DNA of human chromosome 5 hamster hybrid line HyI 90, obtained from Mariano Rocci, Bari (AM409267, AM409266, AM409265) and total human genomic DNA (AM409264, AM409263).
• Randomly subcloned PCR fragments 5IFopt/5IR from pig 9563 have been subcloned in pGem and sequenced.
• the sequences represent 8 portions of satellite sequences within the approximately 341 tandemly arranged dimeric ca. 340 bp units of the human centromere array El (116 kb of the homogeneous alpha satellite array of chromosome 5 belonging to suprachromosomal family I).
• the primers are shown and the positions of the Eco RI restriction sites defining 340 bp dimers are underlined.
• the 8 portions obtained here from pig 9563 are highly similar, but are not identical, which is typical for a random pick up by PCR of some of the up to 341 slightly different units of the entire 116 kb array present in the transferred HAC construct.
• the sequences show that the primers do not only pick up minor variants, and suggest that the human array is indeed stably inherited in the pig supporting authenticity of the El FISH signal on the HAC.
• the sequences also serve re- identification of the centromere proficient El construct, which, after extensive sequencing of random dimers from any source allows isolation of sequences very similar or identical to the 8 sequences shown here.

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