JP2003510072A - Compositions and methods for altering gene expression - Google Patents

Abstract

(57) Abstract Disclosed are novel methods and compositions useful for regulating the expression of a target gene in a cell by introducing an exogenous DNA sequence into the target gene. The compositions and methods of the present invention are useful for producing knockout animals, including mammals. The method for regulating the expression of a eukaryotic gene in a cell according to the present invention comprises transfecting the cell with a nucleic acid construct, the construct comprising a first construct sequence homologous to the first gene sequence, a selectable marker. And a second construct sequence homologous to a second gene sequence, wherein the first and second gene sequences independently comprise at least a portion of one or more intron regions of the eukaryotic gene. And expression of the selectable marker into the mammalian gene, wherein expression of the selectable marker results in regulation of expression of the mammalian gene in the cell.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates generally to biological science, including recombinant genetics and immunology.
More specifically, non-human knockout animals, preferably mammals, are described herein in which the expression of one or more genes is altered. Xenografts are also provided herein in which the expression of one or more genes is regulated to prevent rejection of the graft by the recipient or to reduce the likelihood of rejection. It

BACKGROUND Mammals, including humans, against invading pathogens, toxins, and other foreign substances
Immune response involves many specialized cells that work together. Lymphocytes are a class of white blood cells that are responsible for the specificity of the immune system. Two important classes of lymphocytes are T cells and B cells. T cells develop in the thymus and are responsible for cell-mediated immunity. There are many types of specialized T cells, such as helper T cells (which increase the activity of other types of leukocytes), suppressor T cells (which suppress the activity of other leukocytes). , And cytotoxic T cells, which kill cells. B cells develop in the bone marrow and exert their effect by producing and secreting antibodies.

The secret of a coordinated immune response is complement, which is described in US Pat. No. 5,679,34.
The pathogenesis of tissue damage observed in many immunologically mediated diseases, such as systemic lupus, erythematosis, rheumatoid arthritis, and immuno-hemolytic anemia, as described in No. 5. Get involved in. Complement is also involved in the rejection of transplanted organ transplants. Complement is a tissue injury in a transplant due to an inflammatory condition resulting from rejection or infection in the graft, ischemia, and an inflammatory condition superimposed by vascular thrombosis,
As well as the majority of tissue damage due to inflammation of similar origin in patients who have not received an organ graft. In particular, complement attack of cells is central to the rapid onset of immune-mediated graft rejection (hyperacute rejection), where complement activation and subsequent tissue damage occur within hours.

Graft rejection can occur through many different mechanisms, the time course of rejection being a characteristic of a particular mechanism. Early rejection (hyperacute rejection) that occurs within minutes or hours of transplantation involves complement activation by components present at the time of transplant surgery. Activation is
Via the classical pathway by preformed antibodies reactive with the graft's "foreign" or non-self marker, or as a result of, for example, ischemic damage to organs during pre-transplant storage Can occur via alternative pathways in the response to tissue damage in the implant. Acute rejection occurs days to weeks after transplantation and is caused by sensitization of the host to the foreign tissues that make up the transplant. Once the host's immune system identifies the transplanted tissue as foreign, all means of the immune system are organized against this graft, which is specific (antibody and T cell dependent) response. As well as non-specific (phagocytic and complement dependent) responses. Chronic rejection usually occurs only when the transplant recipient is immunosuppressed. The graft can then survive long enough for the tissue to undergo changes that ultimately affect the survival of the graft. Such changes include hyperplasia and tissue hypertrophy, and endothelial cell damage leading to narrowing of the vessel lumen and strongly reducing the oxygen supply of the graft tissue.

[0005] Porcine tissue xenograft rejection is elicited by natural human antibodies that recognize carbohydrate foreign antigens expressed on porcine endothelial cells that line blood vessels, such as Galα (1,3) galactose. . Weiss, Science, 285 (20)
: 1221-1222 (August 20, 1999). US Pat. No. 5,821,11
No. 7 used the cloned mutant porcine Galα (1,3) galactosyltransferase sequence (specifically, within the exon of the wild-type gene) to clone the wild-type porcine Galα (1,3) galactosyltransferase gene. Inhibition of xenograft rejection by division is described. The resulting mutant gene does not encode a functional galactosyltransferase, consistent with the expected result of avoiding rejection of transplanted xenografts by the patient's immune system.

In such so-called “knockout” mammals, the expression of the endogenous gene is altered (typically suppressed) through genetic engineering. The preparation of knockout mammals typically requires the introduction of nucleic acid constructs into undifferentiated cell types (called embryonic stem cells) in order to repress expression of target genes. The cells are introduced and integrated into the mammalian embryo. The embryo is transferred to a foster mother over the period of pregnancy. For example, Pfeffer et al. (Cell, 73: 457).
-467 [1993]) describes a mouse in which the gene encoding the tumor necrosis factor receptor p55 is disrupted by mutation using homologous recombination. This mouse showed a reduced response to tumor necrosis factor signaling. Fung-L
Eung et al. (Cell, 65: 443-449 [1991]; J. Exp. Me.
d. , 174: 1425-1429 [1991]) describe knockout mice lacking expression of the gene encoding CD8. These mice were found to have reduced levels of cytotoxic T cell responses to various antigens and certain viral pathogens such as lymphocytic choriomeningitis virus.

However, previous exemplary methods describe the manipulation of exon regions of target genes. Therefore, there is a need in the art for new and improved methods for modulating gene expression in animals, including mammals, particularly to overcome xenograft rejection. The following focuses on these and other important objectives.

SUMMARY Expression of a particular gene in an animal introduces a new DNA sequence into the animal's genomic DNA that results in the division of at least some portion of the DNA sequence of the gene to be regulated. It has been discovered that it can be regulated by The methods described herein have general utility for altering gene expression in animals, including mammals. Surprisingly, in contrast to the previous method, the new D
It has been found that by inserting NA sequences into the introns of the target genomic DNA, gene expression can be partially or totally suppressed.

The versatility of the methods described herein for producing “knockout” animals is illustrated by the following general description of preferred embodiments, including the examples. The rest of the discussion relates mostly to the utility of Galα (1,3) galactosyltransferase knockout pigs, but it is understood that the utility of the methods described herein is not limited to just this protein. Should be. Rather, the discussion below is provided merely as an illustration of its versatility and preferred use.

Description of the Preferred Embodiments Technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the described methods, the preferred methods, devices and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing cell lines, vectors and methodologies. These are reported in publications that may be used in connection with this specification.

As used in this specification and the appended claims, the singular form “a”
, "An" and "the" are intended to also include the plural reference forms unless the context clearly dictates otherwise. Thus, for example, reference to "a host cell" is intended to include a plurality of such host cells, "
Reference to "one antibody" is intended to refer to one or more antibodies and their equivalent forms known to those of skill in the art. It should be understood that the appended claims are not limited to the particular methodology, protocols, cell lines, vectors, and reagents described. Those skilled in the art will understand that these can vary. Also, the terminology used herein is for the purpose of describing particular embodiments only and is limited only by the scope of the appended claims.

The term “knockout” refers to the regulation of expression of at least a portion of the protein encoded by the target gene. The term “knockout construct” refers to a nucleic acid sequence designed to regulate the protein encoded by an endogenous DNA sequence in a cell. Nucleic acid sequences used as knockout constructs typically include DNA from a portion of the gene to be regulated (including but not limited to exon sequences, intron sequences, and / or promoter sequences), And a sequence marker used to destroy and select for the knockout construct in the cell, wherein the nucleic acid sequence of the knockout construct is inserted into the cell and interferes or interrupts protein expression from the native gene. Such insertions usually result in homologous recombination (ie, regions of the knockout construct hybridize to the endogenous DNA sequences and to the knockout construct when introduced into the cell). The more soy it is, the more homologous it is Its knockout construct generated recombinantly are) to be incorporated into the corresponding position of the endogenous DNA.

The knockout construct nucleic acid sequence may comprise the full length sequence or subsequence of one or more exons and / or introns of the gene to be regulated, the full length sequence or subsequence of the promoter of the gene to be regulated, or a combination thereof. May be included. In one embodiment of the invention, the nucleic acid sequence of the knockout construct has a first nucleic acid sequence region homologous to a first nucleic acid sequence region of the gene to be regulated and a second nucleic acid sequence of the gene to be regulated. A second nucleic acid sequence region that is homologous to the nucleic acid sequence region of. The orientation of the knockout construct should be such that the first nucleic acid sequence is located upstream of the second nucleic acid sequence, and its sequence marker should be in it.

The appropriate nucleic acid sequence region should be chosen such that there is homology between the knockout construct sequence and the gene of interest. Preferably, the knockout construct sequence is isogenic with respect to the target sequence. The nucleic acid sequence region of the knockout construct can correlate to any region of the gene if it is homologous to the gene. A nucleic acid sequence is "homologous" when it is at least about 90% identical, preferably at least about 95% identical, or most preferably about 100% identical to that nucleic acid sequence. Is regarded as Furthermore, the 5'and 3'nucleic acid sequences flanking the selectable marker should be large enough to provide complementary sequences for hibidization when the knockout construct is introduced into the genomic DNA of the target cell. Is. For example, the homologous nucleic acid sequence flanking the selectable marker should be at least about 500 bp in length,
It should preferably be at least about 1 kilobase (kb), more preferably about 2-4 kb, and most preferably about 3-4 kb. In a preferred embodiment, both homologous nucleic acid sequences flanking the selectable marker of the construct should be at least about 500 bp in length, preferably at least about 1 kb, more preferably about 2-4 kb.
Should be about 3-4 kb, and most preferably about 3-4 kb.

Another suitable DNA sequence comprises a cDNA sequence if the cDNA is insufficiently large. Each of the flanking nucleic acid sequences used to make the construct is preferably homologous to one or more exon and / or intron regions and / or promoter regions. Each of these sequences differ from each other,
It may be homologous to regions within the same exon and / or intron. Alternatively, these sequences may be homologous to regions within different exons and / or introns of the gene. Preferably, the two flanking nucleic acid sequences of the knockout construct are homologous to two sequence regions of the same or different introns of the gene of interest. Furthermore, it is preferred to use isogenic DNA to make the knockout constructs of the invention. Therefore, the nucleic acid sequences obtained for making the knockout constructs are preferably obtained from the same cell line used as the target cells.

According to the invention, the integration of the knockout nucleic acid sequence into at least one gene of interest results in the regulation of the expression of the gene product. "Regulating" the expression of a gene means suppressing the expression of the gene, destroying the expression of the gene, eliminating the expression of the gene, changing the expression of the gene,
Or reducing the expression of that gene relative to the expression of the wild-type gene. Preferably, the integrated knockout construct produces reduced protein function as compared to native protein function. Most preferably, the integrated knockout construct results in production of a non-functional protein. Neither complete nor absolute non-functionality of the protein is required.

The terms “gene disruption” and “gene disruption” refer to the expression of a gene such that the expression of the gene is regulated in the cell as compared to the inherited wild-type or naturally occurring sequence. Refers to the insertion of a nucleic acid sequence into at least one region of the native DNA sequence (usually one or more exons or one or more introns) and / or the promoter region. As an example, the nucleic acid construct may be a DN complementary to the target gene DNA sequence (promoter and / or coding region) to be disrupted.
It can be prepared to contain a DNA sequence encoding an antibiotic resistance gene inserted between A sequences. When the nucleic acid construct is then transfected into cells, the construct is integrated into the genomic DNA either randomly by homologous recombination or in the target gene. Selection for drug resistant cells in a population of transfectants has been found to increase the probability of obtaining a homologous gene knockout. Therefore, many progeny of the cell no longer express the gene or express it at reduced levels. This is because the DNA is now destroyed by the antibiotic resistance gene.

In some cases, production of functional proteins is completely abolished when producing suitable cells, tissues or organs for xenografts in humans, eg, using the methods described herein. It may not be necessary to do. Rather, it is sufficient to reduce the production of functional proteins only to a level that, together with other therapeutic regimens, prevents or reduces the probability of a patient's immune response and rejection. Thus, for example, a knockout performed according to the methods described herein preferably results in a biological activity of the normally encoded polypeptide of at least about 70%, preferably at least 70% relative to the unmutated gene. Can be reduced by about 80%.

The knockout construct can be inserted into any target cell suitable for integration in its genomic DNA, which can be maintained in culture. Suitable cells include, but are not limited to, fibroblasts, epithelial cells, endothelial cells, transgenic embryonic fibroblasts, embryonic stem cells, and primordial germ cells. In one embodiment, the knockout construct is inserted into lung stem cells (ES cells) and integrated into ES cell genomic sequences. ES cells containing the integrated knockout construct are then injected and integrated into the developing mouse embryo. In another implementation, the knockout construct is inserted into a nuclear transfer donor cell. Suitable nuclear transfer donors include: fibroblasts,
Epithelial cells, and cumulus cells. In this embodiment, the knockout construct is inserted into a nuclear transfer donor cell and the donor cell containing the knockout construct is fused with an enucleated oocyte. The resulting fused oocyte is then transferred to a surrogate female. Further, when the target cell is used to produce a knockout mammal, the target cell is preferably derived from the same species as the knockout mammal that is produced. Thus, for example, porcine embryonic stem cells or porcine fibroblasts are commonly used for the production of knockout pigs.

The nucleic acid sequence of the knockout construct can be incorporated into the genomic DNA of the host cell using any suitable method. In one preferred embodiment, the incorporation is
It is achieved by the process of homologous recombination. Homologous recombination has been previously described and is described, for example, in: Kucherlpati et al. (198
4) Proc. Nad. Acad. Sch USA1 1: 3153-3157
Kucherlapati et al .; (1985) Mol. Cell. B
io. 5: 714-720; Smithies et al. (1985) Na
pure 317: 230-234; Wake et al. (1985) Mo
l. Cell. Bio. 8: 2080-2089; Ayares et al.
(1985) Genetics 11 1: 375-388; Ayares e.
t al. (1986) Mol. Cell. Bio. 7: 1656-1662;
Song et al. (1987) Proc. NatI. Acad. Sci.
USA 84: 6820-6824; Thomas et al. (1986)
Cell 44: 419-428; Thomas and Capecchi (1
987) Cell 51: 503-512; Nandi et al. (198
8) Proc. Natl. Acad. Sci. USA 85: 3845-3849.
And Mansour et al. (1988) Nature 336: 3
48-352, which are incorporated herein by reference. further,
Various aspects of using homologous recombination to create specific genetic mutations in embryonic stem cells and transferring these mutations into the germline have been described (Evans).
and Kaufman (1981) Nature 294: 154-14.
6; Doctchinan et al. , (1987) Nature 33
0: 576-578; Thomas and Capecchi (1987) C.
ell 51: 503-512; Thompson et al. (1989)
Cell 56: 316). DN between two DNA molecules in homologous recombination
The A fragment crosses over at sites of homologous nucleic acid sequences.
Exchanged during. Therefore, the crossover is between the knockout construct and the eukaryotic gene at the site of homology within the 5'region of the first nucleic acid sequence of the construct (which is homologous to the first nucleic acid region of the gene of interest). Occurs in. The second crossover event occurs in the 3'region of the construct which is homologous to the second nucleic acid region of the gene of interest. As a result, the sequence information between these two regions of the knockout construct is inserted into the gene of interest in the genomic DNA of the host cell.

The methods described herein can be used to produce a mammal that has knocked out one, two or more genes. Such mammals may be prepared by repeating the procedures set forth herein to produce each knockout construct, or by mating two mammals knocked out of different single genes with each other. , And a mammal with a double knockout genotype.

The term “marker sequence” or “selectable marker” is used as part of a knockout construct that regulates expression of a gene of interest and as a means of identifying those cells that have integrated the knockout construct in their genome. Refers to a nucleic acid sequence.
The selectable marker can be any sequence that serves these purposes. For example, the selectable marker can encode a protein (eg, an antibiotic resistance gene) that confers a detectable property on the cell or an assayable enzyme typically not found on the target cell. These selectable marker genes can be any detectable and / or assayable nucleic acid sequence used to recover the transformed cell line. One of ordinary skill in the art can determine the appropriate selectable marker for use in the present invention. For example, suitable selectable markers include, but are not limited to, β-lactamase (ampicillin resistance), kanamycin resistance, gentacin resistance, puromycin N-acetyl-transferase, hygromycin-β-phosphotransferase, thymidine kinase. , And tryptophan synthetase. For example, herpes simplex virus thymidine kinase (tk) (W
igler, M.I. et al. (1977) Cell 11: 223-32).
Or adenine phosphoribosyl transferase (aprt) (Lowy,
I. et al. (1980) Cell 22: 817-23) genes, which may be used in tk or aprt cells, respectively, may be used as selectable markers. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; eg, dihydrofolate reductase (dhfr) (
This confers resistance to methotrexate) (Wigler, Me.
t al. (1980) Proc. Natl. Acad. Sci. 77: 356
7-70); neomycin phosphotransferase (npt), which confers resistance to aminoglycosides, neomycin and G-418 (Co
lbere-Garapin, F .; et al (I 981) J. Mol
． Biol. 150: 1-14). Additional selectable marker genes have been described and include, for example: tryptophan synthetase (trpB).
(This allows cells to utilize indole instead of tryptophan), or histidinol dehydrogenase (hisD) (which allows cells to utilize histidinol instead of hisgitin) ( Hart
man, S.M. C. and R.D. C. Mulligan (I 988) Pro
c. Natl. Acad. Sci. 85: 8047-51). Recently, the use of visible markers has become popular with markers such as: anthocyanins, β-glucuronidase (GUS) and luciferase and its disposition luciferin. They are not only used extensively to identify transformants, but also to quantify the amount of transient or stable protein expression that can be assigned to a particular vector system (Rhodes , CA et al. (1
995) Methods Mol. Biol. 55.121-131). In the present invention, a selectable marker gene such as an antibiotic resistance gene (eg, neomycin resistance gene or puromycin resistance gene).

Furthermore, if the selectable marker encodes a protein, it may also contain a promoter that regulates its expression or requires expression from an endogenous promoter, preferably a target gene promoter. Thus, the selectable marker may be operably linked to its own promoter or it may be promoterless. The selectable marker gene can be inserted into the knockout construct without its own promoter attached. Because it can be transcribed using the promoter of the gene to be repressed. In addition, the marker gene can have a poly A signal sequence attached to the 3'end of the gene. This signal sequence serves to terminate the transcription of the gene and processes the transcript with the addition of an adenine residue at the 3'end, stabilizing the mRNA.

In one embodiment, the target gene (eg, Galα (1,3) galactosyltransferase) is regulated by the insertion of an engineered exon or active gene within the intron of the target gene. In this embodiment, the target gene is prevented from being translated by insertion of an in-frame promoterless engineered exon (eg, an antibiotic resistance gene) that contains multiple stop codons within the intron of the target gene. It Using this "promoter trap" strategy, engineered exons are spliced in frame upstream of the exons containing the start codon. This results in the expression of the drug resistance gene in front of the gene of interest and at the same time inhibits the expression of the target gene due to the presence of multiple stop codons downstream of the drug resistance gene. As described herein, any gene that confers targeted cell survival under suitable selection conditions can be used as an engineered exon.

A “promoter trap” strategy is used to design gene targeting constructs containing sequences having homology to the intron sequences of the target gene (eg, Galα (1
, 3) Intron 3 sequence of galactosyltransferase gene), a downstream intron splice acceptor signal sequence containing an AG dinucleotide splice acceptor site (for example, Intron 4 splice acceptor signal sequence of Galα (1,3) galactosyltransferase gene). , Exon engineered with a selectable marker without promoter engineered to contain multiple stop codons (eg, drug resistance gene), GT dinucleotide splice donor for splicing exon engineered to an exon immediately downstream A site-containing intron splice donor signal sequence (eg, the intron 4 splice donor sequence of the Galα (1,3) galactosyltransferase gene), as well as the target gene An additional sequence having homology to the intron sequence of the target gene to aid in kneeling (eg, Intron 3 sequence homology of the Galα (1,3) galactosyltransferase gene). It is understood that this method can be used to target any intron within the target gene of interest.

In another embodiment, exons engineered without an in-frame promoter (eg, antibiotic resistance gene) using a “promoter trap” strategy
Target gene expression can be regulated by substituting the endogenous exon in the. The engineered exon is spliced in frame and results in expression of the drug resistance gene, and at the same time inhibits expression of the full length target gene.

This “promoter trap” gene targeting construct contains sequences that have homology to the target gene 3 ′ intron sequence for distillation of the start codon (eg, Galα (1,3)).
A galactosyltransferase gene 3 ′ intron 3 sequence), an upstream intron splice acceptor sequence containing an AG dinucleotide splice acceptor site (eg, intron 3 splice acceptor sequence), a Kozak consensus sequence, eg, poly A termination sequence (engineering) Exon) promoterless selectable marker gene, GT from the intron region downstream of the start codon
It can be designed to include a dinucleotide splice donor site and a splice donor sequence that includes a sequence having 5'sequence homology to a downstream intron (eg, a 5'intron 4 splice donor sequence). It is understood that it can be used to target any exon within the Galα (1,3) galactosyltransferase gene or any other gene of interest. Pig Galα (1,
3) A representative construct useful for targeting the galactosyltransferase gene was published on September 28, 2000, in the American Type Cultu.
re Collection (ATCC), deposited under accession number ___, and described herein in Example 2.

In yet another embodiment, a selectable marker can be inserted into the knockout construct in the opposite orientation to the targeted gene. In this embodiment, strong promoters are used with selectable markers all in the opposite orientation. This drives transcription in the reverse direction and therefore regulates expression of the targeted gene. The target gene is regulated with a "collision construct" to insert the active gene in place of the exon and at least a portion of the flanking introns (including splice donor and splice acceptor sites). The inserted gene (eg, a selectable marker gene) is under the control of a highly active promoter such as the phosphoglycerate kinase I (PGK) gene promoter, so that
Transcription of this gene results in termination of transcription of the endogenous gene (Rosario).
et al. , (1996) Nat. Biotech. 14: 1592-159
6). These selectable marker genes are further engineered to contain transcription termination sequences. An engineered gene insertion can be made to replace any exon or part thereof within any intron, resulting in a truncated transcript that regulates expression of a functional gene product. Positive selection for transfected cells, which have incorporated the construct, can be achieved via expression of a selectable marker gene.
As described herein, it is understood that any selectable marker gene that confers targeted cell survival under suitable selection conditions can be driven by the strong PGK promoter. In addition, the toxin gene (eg, Ricin A toxin) is preferably engineered into a collision construct that has been inserted to eliminate random recombination events. A representative collision construct useful for targeting the porcine Galα (1,3) galactosyltransferase gene is ATCC on September 28, 2000.
Under accession number ____ and is described herein in Example 3.

The selectable marker nucleic acid integrated in the cell can regulate the expression of the gene of interest. Expression of the selectable marker allows for selection of cells containing the integrated sequence. Regulation of expression of the gene of interest is achieved by disrupting the endogenous gene by exons engineered in the same or opposite orientation as the endogenous gene.

The term “animal”, as used herein, is intended to include any multicellular eukaryote, preferably a mammal. When used in the context of a xenograft donor, the term "mammal" preferably includes but is not limited to: pig, sheep, goat, cow, deer, rabbit, hamster, rat, mouse, horse, cat. , Dogs, etc. Preferably humans are excluded.

The term “progeny” refers to any and all future generations that are derived from or originate from a particular mammal (ie, a mammal that contains a knockout construct inserted into its genomic DNA). Thus, the descendants of any consecutive generation are included herein, such that their progeny, such as the F1, F2, F3 generations, etc., are included in this definition for future eternal generation.

The terms “immunomodulatory” and “immunomodulatory” refer to changes in the level of activity of that component of the immune system as compared to the average activity of any component for a particular species. Thus, as used herein, immunomodulation refers to an increase or decrease in activity. Preferably, according to the invention, the incorporation of the selectable marker into the gene of interest results in the phenomenon of an immune response when the host cell is introduced into the patient. Immunomodulation refers to antibody reactivity, complement activation, B cells, T cells of any or all types.
It can be detected by assaying the levels of cells, antigen presenting cells, and any other cells thought to be involved in immune function. In addition, or alternatively, immunomodulation is the level of expression of certain genes that are believed to have a role in the immune system, levels of certain compounds that have a role in the immune system, such as cytokines (such as interleukins) or other molecules. , And / or can be detected by assessing the levels of certain enzymes, proteins, etc. involved in the functioning of the immune system.

The target gene to be knocked out is any gene if at least some sequence information in the DNA to be disrupted is available for use in the preparation of both the knockout construct and the screening probe. obtain.
It is not necessary that the entire genomic sequence of the target gene be known in order to use the methods described herein.

The target gene to be knocked out is a gene expressed in mature and / or immature T cells and / or B cells. It is further preferred that the target gene is expressed in target antigen presenting cells, target endothelial cells, target neurons or any target cells that can be attacked by the recipient's humoral or cellular immune system. This target gene is more preferably involved in the activation pathway during inflammation or immunosuppressive responses by the immune system, either directly or indirectly, and does not cause lethality when knocked out. According to the invention
Expression of the target gene is advantageously modified according to the methods described herein to produce xenograft cells, tissues and organs for use in humans to reduce or prevent immune responses and rejection by patients. be able to.

Therefore, any gene may be used, as long as it can undergo its homologous recombination and expression which can be regulated by the insertion of the knockout construct of the present invention. Suitable genes include, but are not limited to, B7.3, P-selectin,
E-selectin, ICAM-1, ICAM-2 or VCAM-1, CD28,
CD80, CD86, CD154, major compatibility complex class I, β-2-microglobulin, invariant chain (Ii), caspase-1, caspase-3, and Gal.
α (1,3) galactosyl transferase gene. This list is not intended to be exhaustive. One of ordinary skill in the art can identify the appropriate gene to be regulated. Preferably the gene is correlated with the immune response of the patient. More preferably, the target gene is a porcine target gene selected from the group consisting of:
CD80, CD86, B7.3, P-selectin, E-selectin, ICAM-
1, ICAM-2 or VCAM-I. The presently preferred pig target gene is Gal
The α (1,3) galactosyltransferase gene.

The DNA sequence used to knock out the selected gene can be obtained using methods well known in the art as described below: Samb
look et al. (Molecular Cloning: ALabor
atory Manual, Cold Spring Harbor Lab
oratory Press, Cold Spring Harbor, N
． Y. (1989)). Such a method includes, for example, a cDNA encoding at least a part of the same gene in order to obtain at least a part of a genomic sequence.
Screening of genomic libraries with probes. Or c
If the DNA sequence is to be used in a knockout construct, the cDNA can be obtained by screening the cDNA library with an oligonucleotide probe or antibody (if the library has been cloned into an expression vector). If the promoter sequence is to be used in a knockout construct, synthetic DNA probes can be designed to screen a genomic library containing the promoter sequence. Another way to obtain the DNA to be used in the knockout construct is to synthetically produce the DNA sequence using a DNA synthesizer.

In another embodiment, porcine genomic DNA encoding the Galα (1,3) galactosyltransferase gene is isolated from a λ phage clone library. The porcine genomic library is screened with the cDNA corresponding to exon 4 of the Galα (1,3) galactosyltransferase gene. Phages are screened and unique clones containing exon 4 sequences are isolated using standard library screening methods (Sam.
Brook et al. ). Clones obtained using this procedure contain inserts 15-40 kb long. These clones are pgGT, λ1, λ2, λ4
-1 and λ8-2. Pig G from intron 3 to intron 8
The following five vectors containing unique overlapping nucleotide sequences throughout the alα (1,3) galactosyltransferase gene have been deposited with the ATCC: (1) The 5'most of the 18.275 kb λ-2 phage clone. 1.6 kb within terminal intron 3, (2) 6.7 kb HindIII fragment spanning intron 3 to intron 4 of 18.275 kb λ-2 phage clone; (3) 6.7 kb of 18.275 kb λ-2 phage clone. A 4 kb HindIII fragment after the fragment, (4) a 6 kb HindIII-SalI fragment at the 3 ′ extreme end of the 18.275 λ-2 phage clone, and (5) a λ-2 phage clone spanning exon 7 to exon 9. 13 kb fragment of These five vectors were added to ATCC 2000
They were deposited on September 29, 2014 with the following deposit numbers ___. Subclones of the various inserts were used to generate the claimed intron sequences from intron 3 to intron 8, as provided in FIG. Using these sequences, pig G
Regions of sequence homology can be determined in the design targeting construct for the regulation of the alα (1,3) galactosyltransferase gene.

The DNA sequence encoding the knockout construct is preferably produced in sufficient quantity for genetically engineering and inserting the target cell. Propagation can be achieved by known methods such as: inserting the sequence into an appropriate vector and transforming a bacterial cell or other cell capable of rapidly growing the vector,
By PCR amplification or synthesis by a DNA synthesizer.

The DNA sequence to be used in producing the knockout construct is digested with the restriction enzymes selected to cut the position so that the new DNA sequence encoding the selectable marker gene is within this DNA sequence. Can be inserted at the appropriate position. Appropriate positions for insertion of the selectable marker gene are those that serve to regulate expression of the native gene. This position depends on the restriction sites in the sequence to be cleaved and various factors such as whether intron, exon or promoter sequences are to be regulated. In other words, the precise location of insertion of the selectable marker into its DNA sequence results in regulation of promoter function or synthesis of the native exon. For example, the knockout construct can be engineered to insert an entire selectable marker within a single intron of the target gene. Thus, the first nucleic acid sequence comprises a region of the selected intron upstream from the second nucleic acid sequence, the second nucleic acid sequence being selected downstream of the first nucleic acid sequence. Selected to include regions of the intron. The selectable marker is introduced between the first and second nucleic acid sequences. Then, when the construct is introduced into the cell, the construct nucleic acid sequence is integrated into the target gene and the selectable marker is entirely inserted within the targeted intron.

Similarly, the construct can be engineered to insert a selectable marker within any desired and appropriate region of the gene, provided expression is regulated. For example, the construct inserts a selectable marker between two flanking introns, thereby removing at least a portion of the target gene intron and at least a portion of the flanking intron so as to completely eliminate the endogenous exon of the target gene. To span a region containing at least a portion of an intron flanking a promoter for a target gene, span a region containing more than one target gene, and combinations thereof. Can be manipulated.

After digesting the genomic DNA sequence with the appropriate restriction enzymes, the selectable marker gene is ligated to the genomic DNA sequence using methods well known to those of skill in the art and those described by Sambrook et al., Supra. The ends of the DNA fragments to be ligated must be compatible; this can either be cleavage of all fragments with an enzyme that produces compatible ends, or blunting of the ends prior to ligation. Achieved by Blunting is performed using methods well known in the art, for example by using Klenow fragment (DNA polymerase I) to fill in the sticky ends.

The ligated knockout construct can be introduced directly into the target cell or first placed in a suitable vector for amplification prior to insertion. The preferred vector is
pBluescript II SK vector (Stratagen, San
Diego, Calif. ) Or pGEM7 (Promega Corp.,
Madison, Wis. ), Which is rapidly amplified in bacterial cells.

In another embodiment of the invention, embryonic stem (ES) cells are used as target cells. This is due to its ability to integrate into and become part of the germline of the developing embryo, resulting in germline transmission of the knockout construct. Therefore, any ES cell line that is believed to have this ability is suitable for use in the present invention. For example, one mouse strain used to generate ES cells is the 129J strain. These cells are cultivated and prepared for DNA insertion using methods well known to those of skill in the art. These methods are described, for example, in Robertson (Ter
atocarcinomas and Embryonic Stem Cel
ls: A Practical Approach, E.I. J. Robertso
n, IRL Press. Washington, DC (1987)), Br
adley et al. (Current Topics in Dev. Biol.
, 20: 357-371 (1986)), and Hogan et al. (Manipulu).
eating the Mouse Embryo: A Laboratory
Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.M. Y. (1986)
), All of which are incorporated herein by reference.

Insertion of the knockout construct into target cells can be accomplished using various transfection methods well known in the art. For example, suitable transfection methods include electroporation, microinjection and calcium phosphate treatment (see Lovell-Badge, Robertson, Ed., Supra). The preferred transfection method is electroporation. When subjecting the cells to electroporation, the target cells and knockout construct DNA are exposed to an electrical pulse using an electroporation device and according to the manufacturer's guidelines for its use. After electroporation, cells can be harvested under appropriate incubation conditions. The cells are then screened for the presence of this knockout construct.

Each knockout construct DNA introduced into the cell must first be linearized if the knockout construct is inserted into the vector. The linearization is carried out with the appropriate restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.
Is achieved by digesting.

For the introduction of the DNA sequence, the knockout construct DNA is added to target cells under conditions suitable for the chosen insertion method. When more than one construct is introduced into target cells, the DNA encoding each construct is
Can be introduced one by one.

Screening can be performed using methods known in the art or combinations thereof. If the selectable marker gene is an antibiotic resistance gene, the cells are otherwise cultured in the presence of lethal concentrations of antibiotic. The surviving cells are probably
It incorporates this knockout construct. When the selectable marker gene is other than the antibiotic resistance gene, the genomic DNA of the target cell is analyzed by standard methods (for example, S
The methods described in ambrook et al., supra) can be used to extract from those cells. This DNA can then be probed on Southern blots with probe (s) designed to hybridize only to its selectable marker sequence. If the selectable marker gene is a gene encoding an enzyme capable of detecting activity (eg, β-galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and a suitable assay for enzymatic activity can be performed. Can be done. In addition, genomic DNA may be amplified by the polymerase chain reaction (PCR) using probes specifically designed to amplify DNA fragments of specific size and sequence (ie, containing this knockout construct at the appropriate position). Only cells produce DNA fragments of the appropriate size). PCR can be used in the detection of the presence of homologous recombination (Kim and Smithies, (1988).
Nucleic Acid Res. 16: 8887-8903: Joyner.
(1989) Nature 338: 153-157). Primers complementary to sequences within the construct and primers outside the construct and complementary to sequences at the target locus can be used. In this way, only DNA duplexes with both primers present in the complementary strand where homologous recombination has occurred can be obtained. The occurrence of homologous recombination is supported by demonstrating the presence of primer sequences or sequences of the expected size.

A double crossover (Do) is provided upstream and / or downstream of the target gene knockout construct.
A gene may be inserted to identify whether a double crossover has occurred. For this purpose, any suitable marker can be used as described herein. Preferably, the selectable marker used to identify the double crossover is different than the selectable marker used to identify the integration of the target gene knockout construct. In one preferred embodiment, the herpes simplex virus thymidine kinase gene is used. This is because the presence of this thymidine kinase gene can be detected using nucleoside analogs (eg acyclovir or ganciclovir) by their cytotoxic effect on cells containing a functional HSV-tk gene. The absence of sensitivity to these nucleoside analogs indicates the absence of this thymidine kinase gene, and thus, when homologous recombination occurs, a double crossover event also occurs.

Knockout constructs can integrate at several positions in the target gene genome and at different positions in each cellular genome due to the occurrence of random insertion events. For random multiple integration sites, the desired insertion site is at the position complementary to the DNA sequence being knocked out. It has been found that less than about 1-5% of the targeted cells that take up the knockout construct actually incorporate the knockout construct at the desired location. Identification of cells with proper integration of the knockout construct is described herein.

In one embodiment of the invention, appropriately transfected target cells containing the knockout construct in its appropriate position are inserted into the embryo. Insertion can be accomplished in any suitable manner known in the art. Preferably, the cells are introduced into the embryo by microinjection. Most preferably, the cells are ES cells for injection into mouse embryos. For microinjection,
About 10-30 cells are collected in a micropipette and injected into embryos at the appropriate developmental stage to incorporate the transfected cells into the developing embryo. The appropriate stage of development for injection into the embryo is the germ of the developing embryo.
before the formation of the inner layer), which can be easily determined by those skilled in the art.
Preferably, the embryo is in the early blastocyst stage. For example, mouse embryos can be transfected with the transfected cells in about 3.5 days. Embryos are obtained by perfusing the uterus of a pregnant female by methods known to those skilled in the art (eg Bradley (Robertson, supra)). Preferably, the embryo is male.

After introducing the transfected target cells with the appropriate integration of the target gene into the embryo, the embryo is transplanted into the uterus of a pseudo-pregnant mother. Although any mother-in-law can be used, the mother-in-law's choice is based on its ability to grow and reproduce well and to care for its young. Such mother-in-law may typically be made by mating with a vasectomized male of the same species. The pseudopregnant mother-in-law stage is important for successful transplantation and is species-dependent. For mice, this stage is about 2-3 days pseudo-pregnancy.

In another embodiment, the suitable, transfected cells are nuclear transfer donor cells. Nuclear transfer donor cells can be virtually any somatic cell type, including fibroblasts, epithelial cells, cumulus cells and the like. Nuclear transfer donor cells are cultured in vitro and targeted via homologous recombination using the constructs and techniques described herein. Cells
Allows selection of cells grown in the appropriate medium and containing the properly integrated knockout construct. PCR can also be performed for confirmation of correctly targeted integration. After this, the unfertilized oocytes of the animals are enucleated using known methods. The enucleated, unfertilized oocytes are then fused to the selected knockout nuclear transfer donor cells. Fusion can be done by electrical stimulation, chemical stimulation, insertion by injection, or other known methods. The fused product is then cultured, evaluated for viability, and transferred to surrogate recipient females. For references and methods, see, for example, Campbell et al. (1996) Nature 380: 64; W.
ilmut et al. (1997) Nature 385: 810; WO00 / 2557.
8; WO97 / 07669; WO99 / 36510; WO00 / 42174; W
See O99 / 53751; WO99 / 45100, which are incorporated herein by reference.

Offspring born from this mother-in-law or surrogate recipient female
Or a progeny of genomic DNA containing this knockout construct
(For example, by PCR). This step is particularly important for selecting offspring of mother-in-law carrying embryos injected with this transfected target cell. In contrast, the offspring of the surrogate recipient female carrying this transfected target cell fused with an enucleated unfertilized oocyte typically has the knockout construct inserted within its genome.

Any suitable selection method may be used. For example, coat color
3.) If a selection strategy is used, the progeny may be screened for coat color indicating proper integration of the targeted gene into the progeny. Other methods include obtaining DNA from progeny and screening for the presence of this knockout construct using Southern blots and / or PCR, as described herein. Other means of identifying and characterizing knockout progeny include the use of Northern and Western blots. For example, Northern blots can be used to determine the mR for the presence or absence of a transcript encoding a knocked out gene, a marker gene or both.
NA can be probed. In addition, Western blots will be used to assess the level of expression of the knocked out gene in various tissues of these progeny.
This is by probing the Western blot with an antibody to the protein encoded by the knocked out gene or an antibody to the marker gene product (if the gene is expressed). In situ analysis of various cells from progeny (eg, fixing cells and labeling with antibodies) and / or fluorescence activated cell separation (FACS) analysis to confirm the presence or absence of this knockout construct gene product. This can be done using the appropriate antibody.

If the offspring that are shown to contain the integrated knockout construct in their genome then carry the knockout construct in their germline and are considered to make F1 progeny heterozygous for this knockout construct. , The offspring can be out-crossed to produce multiple offspring. F1 is then mated to create homozygous animals.

The heterozygotes are then bred to each other to create homozygous knockout progeny. Homozygotes can be identified by any screening method as described herein. For example, homozygotes are host animals that are the product of this mating,
And known heterozygotes and wild-type host animals can be identified by Southern blot of equivalent amounts of genomic DNA. Probes for screening Southern blots can be designed as shown herein.

The knockout mammals described herein have a variety of uses, depending on the gene (s) that are regulated. If the regulated targeted gene (s) encode proteins thought to be involved in this immunosuppression or inflammation, knockout mammals will be used to identify drugs useful in immunomodulation (ie Drug) that enhances or inhibits activity). Screening for useful drugs involves administering a candidate drug to knockout animals over a range of dosages and assaying the drug's immunomodulatory effect on the immune disorder being evaluated at various times. Such assays include, for example, increasing or decreasing T and B cell levels, increasing or decreasing immunoglobulin production, increasing or decreasing levels of chemical messengers (eg, cytokines such as interleukins), and And / or confirming an increase or decrease in the expression level of a particular gene involved in the immune response.

For example, patients undergoing chemotherapy often undergo immunosuppression. It is desirable to activate the immune system of such individuals, by administering to the patient a therapeutic agent that can produce such effects. Knockout animals as described herein are screened for various compounds (alone or in combination) to determine whether partial or total repair or activation of the immune response occurs. Can be used for.

Similarly, applying the same strategy would be useful in suppressing the inflammatory response observed in many patients with arthritis, or of the autoimmune phenomenon observed in patients with rheumatoid arthritis and lupus. One may find compounds that are useful for inhibition. In addition, mammals may be useful in assessing the development of the immune system and studying the effects of certain genetic mutations.

In a preferred embodiment, the knockout mammals described herein are used for xenografts in human patients. The xenograft tissue can be from any mammal, preferably a pig. Xenograft tissue can be, for example, kidney, heart, lung,
Or it may be in the form of an organ, including the liver. Xenograft tissue can also be in the form of organs, cell populations, and parts of glands, including, for example, lenses, islet cells, skin, corneas, and the like.

In another aspect of the invention, the target gene is porcine Galα (1,3) galactosyltransferase. Galα (1,3) galactosyltransferase is an attractive target for knockout in pigs. This enzyme is responsible for the addition of carbohydrate residues (Galα (1,3) Gal). Galα (1,
3) Gal is a human IgM antibody and IgG in pig vs human xenograft tissue.
It is recognized by the antibody and subsequently leads to hyperacute rejection. Therefore, knockout pigs, which lack the Galα (1,3) galactosyltransferase gene, may serve as a potential source as a rich source for transplant organs.
Disclosed are nucleic acid sequences encoding Galα (1,3) galactosyltransferase and variants thereof. Preferably, this nucleotide sequence is porcine Gal
It encodes α (1,3) galactosyltransferase. The nucleotide sequence can be in the form of DNA, RNA, or a mixture thereof. The nucleotide sequence or isolated nucleic acid can be inserted into replicating DNA, RNA or DNA / RNA vectors (well known to those skilled in the art) (eg, plasmids, viral vectors, etc.) (Sambrooks et al., Molecular Cloning: A).
Laboratory Manual, Cold Spring Harbor
Laboratory Pess, NY. 2nd edition, 1989).

The nucleotide sequence encoding Galα (1,3) galactosyltransferase can include promoters, enhancers, and other regulatory sequences for expression, transcription and translation. Vectors encoding such sequences may contain additional genes and / or selectable markers, as well as restriction enzyme sites for insertion of elements necessary for growth and maintenance of the vector in cells.

Targeting constructs comprising the nucleotide sequence of Galα (1,3) galactosyltransferase and variants thereof, which inactivate the wild-type Galα (1,3) galactosyltransferase gene according to the method of the invention. Is particularly preferable when it can be used for. Variant Galα (1,3) galactosyltransferase nucleotide sequences include, but are not limited to, nucleotide deletions, insertions, substitutions and additions relative to wild-type Galα (1,3) galactosyltransferase. As a result, the resulting mutant does not encode a functional galactosyltransferase. These nucleotide sequences can be used in a method of regulating the expression of the galactosyltransferase of the present invention. In this manner, the variant sequence can recombine with the wild-type genomic sequence in the target cell.

In the most preferred embodiment, a knockout pig is generated in which the Galα (1,3) galactosyltransferase gene produces a non-functional protein. Human antibodies that produce a non-functional protein that bind to the Galα (1,3) Gal epitope otherwise expressed in xenograft tissue do not bind, resulting in tissue rejection. prevent. In this embodiment, any knockout construct that can modulate the interaction between the antibodies for Galα (1,3) galactosyltransferase binding can be used.

EXAMPLES The following examples are for purposes of illustration only and are not intended to limit the scope of the disclosure or claims herein.

Example 1 Inactivation of the Galα (1,3) Galactosyltransferase Gene by Inserting an Engineered Active Gene in the Form of an Engineered Exon in Intron 3 In this example, Galα (1,3) 3) Prevent the galactosyl transferase protein from being translated by the in-frame insertion of engineered exons lacking a promoter (eg, antibiotic resistance genes). Engineered exons lacking this promoter contain multiple stop codons within the intron of the Galα (1,3) galactosyltransferase gene. Using this "promoter-trap" strategy, the engineered exon is spliced in frame upstream of exon 4 of the Galα (1,3) galactosyltransferase gene. This causes the expression of the drug resistance gene to precede the gene of interest and simultaneously inhibits the expression of the transferase gene due to the presence of multiple stop codons downstream of the drug resistance gene. As described herein, any gene that confers target cell survival under suitable selection conditions may be used as an engineered exon, including, but not limited to: ampicillin, kanamycin, Geneticin, neomycin phosphotransferase (
phosphophospherase), puromycin N-acetyltransferase, hygromycin b-phosphotransferase, thymidine kinase, and tryptophan synthetase. This example uses neomycin.

Design gene targeting constructs. The gene targeting construct is G
alα (1,3) galactosyltransferase gene 5 ′ intron 3 sequence, intron 4 splice acceptor signal sequence, neomycin phosphotransferase gene (engineered exon) lacking a promoter genetically engineered to contain multiple stop codons, exon 4 Containing a sequence with homology to an intron 4 splice donor sequence for splicing an engineered exon downstream of, and an additional intron 3 sequence homology to assist annealing to the porcine Galα (1,3) galactosyltransferase gene . Although this example describes targeting intron 3, the method can be used to target any intron within the Galα (1,3) galactosyltransferase gene or any other gene of interest. Galα (1,3
) A sequence listing of the introns (from within intron 3 to the end of intron 8) in the galactosyltransferase gene is provided in Figure 1. Schematic diagrams of targeting vectors and corresponding nucleotide sequences are shown in FIGS. 2 and 3.

The gene targeting construct was divided into five separate DNA fragments (1 below).
-5) are ligated together to form the final gene targeting construct using standard molecular biology techniques well known to those of skill in the art. The PCR reaction was performed according to the manufacturer's instructions on ELONGASE Enzyme Mix (Life).
(Technologies, Gathersburg, MD). In this example, 50 μl final reaction volume (2 μl DNA template, 1 μl E
LONGATE Enzyme Mix, 60 mM Tris-SO ₄ (pH 9
． 1), 18 mM (NH ₄ ) ₂ SO ₄ , 1.2 mM MgSO ₄ , 200 mM d
NTP mix, with 10% DMSO and 200 mM of each primer). This reaction was run on a standard PCR thermocycler (GenAmp PCR).
System 2400; PE Applied Biosystems, F.
hot start at 95 ° C for 1 minute in
Then, 30 to 40 cycles are performed.

1. A polymerase chain reaction (PCR) product containing the intron 3 sequences listed in Figures 1 and 3 (nucleotide numbers 10-4020) is generated using standard PCR conditions for long range PCR of genomic fragments. . The primers used were NotI restriction site and intron 3 sequence 1
5'primer containing 0-23

[0070]

[Chemical 1] And a 3'primer containing a SalI restriction site and a sequence homologous to the intron 3 sequence 3999-4020

[0071]

[Chemical 2] , Where the underlined sequences indicate restriction sites and the bold letters indicate homology to the exogenous sequence. An additional guanine nucleotide was added to 5 of all probes in this example to match the 1 bp deletion that occasionally occurs during cloning.
'Add to the end.

2. The PCR product was transformed into an intron 4, 3 ′ splice sequence (pyrimidine-rich lasso structure and Galα (1,3) galactosyltransferase trontron 3 dinucleotide acceptor sequence), and 196 bases of 5 ′ flanking ag dinucleotide. Site (nucleotide 11521 in FIGS. 1 and 3)
-11716) is included. The primer used was a 5'primer containing a SalI restriction site.

[0073]

[Chemical 3] ; And EcoRI restriction sites and pyrimidine-rich lasso structure and Gal
3'primer containing complementary strand homologous to α (1,3) galactosyltransferase dinucleotide acceptor sequence

[0074]

[Chemical 4] , Where the underlined sequences indicate restriction sites and the bold type indicates sequences that have homology to the exogenous sequence.

3. Neomycin resistance gene (GenBank accession number # AF081957)
5'primer containing an EcoRI restriction site containing the ATG start codon and homology to the neomycin resistance gene;

[0076]

[Chemical 5] A 3'primer comprising a HindIII restriction site and a natural stop codon, followed by a complementary sequence to the 3'codon region of the neomycin gene containing two additional engineered stop codons;

[0077]

[Chemical 6] To generate. Here, underlined sequences indicate restriction sites, and bold letters indicate sequences with homology to the exogenous sequence (see Figures 3 and 4).
.

4. The PCR product was loaded with the 5'splicing donor sequence for intron 4 of the Galα (1,3) galactosyl transferase gene, which corresponds to sequences 4938-5173 (FIGS. 1 and 3) of the present sequence containing intron 4. Generate to include. The primers used were 5 ′ containing a sequence identical to intron 4, sequences 4938-4962, containing a HindIII restriction site and a Galα (1,3) galactosyltransferase dinucleotide splice site.
Primer

[0079]

[Chemical 7] And a PstI site and a complementary strand sequence from intron 4 corresponding to nucleotide numbers 5152-5173 and comprising multiple stop codons.

[0080]

[Chemical 8] , Where underlined sequences indicate restriction sites, and bold type indicates sequences with homology to exogenous sequences.

5. The PCR product containing 1150 nucleotides of intron 3 corresponds to nucleotides 4024 to 4826 (FIGS. 1 and 3) of the sequence of the present application. The primer used was 5'containing a PstI site and sequences 4024-4050 of the sequence of the present application.
Primer

[0082]

[Chemical 9] And a 3'primer containing an XhoI restriction site and a complementary strand sequence to sequences 4801 to 4826 of the present application.

[0083]

[Chemical 10] , Where underlined sequences indicate restriction sites, and bold type indicates sequences with homology to exogenous sequences.

Each PCR fragment (steps 1-5) is amplified respectively. The single PCR fragment is cloned into the pCR2.1 vector (Invitrogen, San Diego, CA) according to the manufacturer's ligation instructions.
Recombinant plasmid DNA was transformed into a suitable bacterial host (Invitrogen, San
(Diego, CA). The bacterium was cultivated and plasmid D
NA is isolated. Plasmid DNA with the correct insert (determined by restriction and sequence analysis) is used to construct the final product.

Following PCR fragment amplification, a series of ligations is performed to clone the final construct in the bacterial plasmid pBS SK + (Stratagene, La Jolla, CA).

A. The HindIII-PstI fragment from the intron 4 PCR product (step 4) and the PstI-XhoI 3 ′ homology fragment from intron 3 (step 5 above) were ligated into pBS SK + vector DNA and then Hind.
Digest with III and XhoI. The three DNA fragments were mixed in an equimolar ratio and according to the manufacturer's recommendations, T4 DNA ligase (New E).
Incubation in the presence of ngland Biolabs, Beverly, MA). After ligation, the recombinant plasmid DNA was transformed into a suitable bacterial host (D
H10B, Life Technologies, Geithersburg,
MD). Bacteria are cultivated and plasmid DNA is isolated. A plasmid with the correct insert (determined by restriction and sequence analysis)
Is used to build the final product.

B. The obtained plasmid (step 5a) was treated with HindIII and EcoRI.
Digested with and ligated with the HindIII-EcoRI neomycin resistance gene fragment (step 3), which was previously digested with HindIII and EcoRI. The resulting recombinant plasmid DNA was transformed into a suitable bacterial host (DH10B, Life Technologies, Geithersbur.
g, MD). Bacteria are cultivated and plasmid DNA is isolated. The plasmid with the correct insert (as determined by restriction and sequence analysis) is used to construct the final product.

C. The resulting plasmid (step 5b) was digested with EcoRI and NotI and the SalI-EcoRI intron 4-3 ′ splicing fragment (step 2) (which was previously digested with SalI and EcoRI and the intron The 34 kb NotI-salI fragment (step 1) is N
digested with otI and SalI). The three DNA fragments were added to the T4 DNA ligase (in equimolar ratio according to the manufacturer's recommendations).
Incubate in the presence of New England Biolabs, Beverly, MA). After ligation, the recombinant plasmid DNA was transformed into a suitable bacterial host (DH10B, Life Technologies, Geithers).
burg, MD). Bacteria are cultivated and recombinant plasmid DN
Isolate A.

The final construct is used to transfect porcine embryonic fibroblasts, transgenic porcine fibroblasts, or porcine embryonic stem cells, or porcine primordial germ cells.
Cell clones resistant to neomycin were screened for PC to determine the site of integration.
Screen by R. Primers (complementary strands of 5407 to 5427; GGACAATGGCA) arranged in a region of intron 4 which is not incorporated into the final construct.
ACATGGCAGG; see Figures 1 and 3) is used in combination with the 5'neomycin gene primer (step 3). Only the targeted insert will yield a PCR fragment of the appropriate size. All other integration events give negative results.

The cell clone with the targeted insert is then used to generate transgenic animals using nuclear transfer technology (or in the case of stem cells) and injected into developing undifferentiated embryo cells. , And produce chimeric offspring.

Example 2 Gal by Substitution of Exon 4 with an Engineered Exon Form of the Active Gene
Inactivation of α (1,3) galactosyltransferase gene In this example, Galα (1,3) galactosyltransferase protein was engineered exon (antibiotic) in which the exogenous exon (exon 3) was lacking a promoter in frame. It was prevented from being translated by exchanging it with a resistance gene). This engineered exon contained a bovine growth hormone polyA sequence attached to the 3'end of the gene, which served to terminate transcription of the engineered exon. The engineered exons were spliced in-frame to take advantage of the exogenous promoter typically used by the Galα (1,3) galactosyltransferase gene (“promoter-trap” strategy). This results in the expression of drug resistance genes and, at the same time, full-length Ga
The expression of the lα (1,3) galactosyltransferase gene was inhibited. As described herein, any gene that confers target cell survival under suitable selection conditions may be used as an engineered exon, including, but not limited to: ampicillin, kanamycin, Geneticin,
Neomycin phosphotransferase
as)), puromycin N-acetyltransferase, hygromycin b
-Phosphotransferases, thymidine kinases, and tryptophan synthetases. This example utilizes puromycin.

Galα (1,3) galactosyltransferase gene 3 ′ intron 3
Sequence, a sequence having homology to intron 3 splice signal sequence (splice acceptor sequence), Kozak consensus sequence, bovine growth hormone poly A sequence (b poly A) (engineered exon), promoterless puromycin A gene targeting construct was designed containing the N-acetyltransferase gene, a 5'intron 4 splice signal sequence (splice donor sequence) and a sequence with 5'intron 4 sequence homology. Exon 4 of the Galα (1,3) galactosyltransferase gene encodes the ATG start codon and the N-terminal part of this protein. This example describes targeting introns 3 and 4, but this method is for targeting any exon within the Galα (1,3) galactosyltransferase gene or any other gene of interest. It is anticipated that it could be used. A sequence listing of the introns of the Galα (1,3) galactosyltransferase gene (from within intron 3 to the end of intron 8) is provided in FIG.

The gene targeting construct was constructed using standard molecular biology techniques well known to those skilled in the art.
It was generated by joining two different DNA fragments together to form the final gene targeting construct. The first DNA fragment was obtained from the 3'end of intron 3 containing the 3'splice sequence, a pyrimidine-rich branch site and an AG dinucleotide splice acceptor sequence used in forming the lasso structure during splicing. It was The second DNA fragment was obtained from the 5'end of intron 4 containing the AT dinucleotide splice donor sequence. This fragment was ligated into the pBluescript vector containing the Kozak consensus sequence in frame with the coding sequence for the promoterless puromycin gene linked to the bovine growth hormone poly A sequence (Figure 5) to form the final gene construct. Schematics of targeting vectors and corresponding nucleotide sequences are shown in FIGS. 6 and 7.
Shown in. In addition, this construct was deposited with the ATCC on September 28, 2000 Deposited at.

(1. Preparation of First DNA Fragment) The first DNA fragment is a polymerase chain reaction (PCR) product consisting of the intron 3 sequence shown in FIG. 7 (nucleotide numbers 235 to 4851 (from lambda phage clone) (For position 1 of the nucleotides of the isolated insert), and for long-range PCR of the genomic fragment, using standard PCR conditions as described by Randolf et al. (1996). The 5'primer consisting of 3 sequences 235 to 260 is 5'-AAGATT.
ATAAATAGCCTCTCGTTCAGG-3 '. The 3'reverse primer sequence is complementary to sequences 4827-4851 at the most 3'end of intron 3, and contains an AG splice acceptor site, and is 5'-CTC.
It was CTGGGAAAAGAAAAGGAGAAGG-3 '.

The PCR reaction conditions for producing the 4.616 kb intron 3 sequence were EL
ONGASE Enzyme Mix (Life Technologies,
Gaithersburg, MD) according to the manufacturer's conditions. In this example, 2 μl DNA template, 1 μl ELONGASE E
nzyme Mix, 60 mM Tris SO ₄ (pH 9.1), 18 mM (NH ₄ ) ₂ SO ₄ , 1.2 mM MgSO ₄ , 200 mM dNTP mixture, 10
A final reaction volume of 50 μl was used with% DMSO and 200 nM of each primer. The reaction was hot-started at 95 ° C for 1 minute, followed by standard PC
Performed 30-40 cycles in the R apparatus (eg, Gene Amp PCT S
systems 2400: PE Applied Biosystems, Fo
(Ster City, CA).

(2. Preparation of PCR2.1 Cloning Vector) PCR2.1 Cloning Vector (Invitrogen, San Die)
The NotI site of go, CA) was destroyed to prevent it from becoming the final construct over a second NotI site. The NotI site is unique and was used to linearize the final plasmid construct. The PCR2.1 vector was digested with NotI and the Klenow enzyme (Roche Molecular Bioche).
the overhangs were filled in according to the manufacturer's specifications. The plasmids were religated with T4 DNA ligase (New England Biolabs, Beverly, MA) according to the manufacturer's recommendations. The plasmid DNA was transformed into a suitable bacterial host (Top 10F ', Invitrogen, Dan Diego, CA). The bacterium is cultivated and the plasmid DNA is isolated and
Incubation with the otI enzyme confirmed the loss of this site by restriction analysis.

(3. Insertion of First DNA Fragment into PCR2.1 Vector) Following PCR, the 4.616 kb fragment was modified with T4 DNA ligase according to the manufacturer's specifications to prepare the modified PCR2.1 vector. Combined with. Plasmid DNA was transformed into a suitable bacterial host (Top 10F ', Invitrogen, D
an Diego, CA). The bacterium was cultivated and plasmid DNA was isolated. To construct the final product, a plasmid with the correct insert in the proper orientation, as determined by restriction and sequence analysis genes, was used.

4. Preparation of Second DNA Fragment A 2.084 kbp PCR product consisting of an intron 4 homology sequence containing the GT dinucleotide acceptor consensus splice sequence was added to a standard PCR product as described in step 1 above. Constructed using PCR conditions. The 5'primer consisting of the sequences 4938 to 4961 at the most 5'end of intron 4 is 5'-GTAATTATGA.
It was AACATGATGAAAATG-3 '. The 3'primer is 6997-
It is homologous to the complementary strand of intron 4 at position 7021 and has the sequence 5'AGC.
CAGCGCTTAACTAAGTACGTTGC-3 '.

(5. Insertion of Second DNA Fragment into PCR2.1 Vector) Following PCR, a 2.084 kb fragment was inserted into Invitrogen (S
ligation into the pCR2.1 vector from An Diego, CA) using the manufacturer's coupling conditions. After ligation, the recombinant plasmid DNA is transformed into a suitable bacterial host (T
op 10F ', Invitrogen, Dan Diego, CA)). The bacterium was cultivated and plasmid DNA was isolated. To construct the final product, as determined by restriction analysis and sequence analysis genes,
A plasmid with the correct insertion and orientation within the plasmid was used.

6. Preparation of Synthetic Oligonucleotide Linker Sequences Synthetic oligonucleotide linkers containing a Kozak consensus sequence and associated restriction enzyme sites were prepared for the following in-frame cloning of the promoterless puromycin gene.

[0101]

[Chemical 11] (7. Assembly of gene targeting construct)
The final construct was made in La Jolla, CA. This final construct
An example is shown in FIG.

A. Oligonucleotide linker containing Kozak consensus sequence (step 6)
Was ligated to KS + vector DNA after digestion with XhoI and EcoRI. The ligation was performed using T4 DNA ligase (New England Biol).
Abs, Beverly, MA) using linker: vector in a molar ratio of at least 3: 1 according to the manufacturer's recommendations. After ligation, the recombinant plasmid was transformed into a suitable bacterial host (XL1-Blue MRF ', Strategene,
La Jolla, CA). The bacterium was cultivated and plasmid DNA was isolated. Unique restriction sites within the linker (BglII or Hpa
Restriction enzyme analysis was performed with I) to confirm successful binding. This plasmid containing a linker was then used to construct the final product.

B. The resulting "mother" plasmid (step 7a) was then digested with EcoRV and SpeI and cloned in the 3'arm of the targeting construct. P
The 2.804 kb PCR fragment cloned into the CR2.1 vector (step 5) was digested with EcoRV and SpeI, isolated from vector DNA by agarose gel electrophoresis and purified. The 2.804 kb fragment was ligated between the EcoRV and SpeI sites of the mother plasmid (step 7a). The ligation was carried out according to the manufacturer's recommendations, T4 DNA ligase (New E).
ng Biolabs, Beverly, MA) with a 3: 1 molar ratio of insert: vector. After ligation, the recombinant plasmid was transformed into a suitable bacterial host (XL1-Blue MRF ', Stratgen, La Jol.
la, CA). The bacterium was cultivated and plasmid DNA was isolated. The plasmid with the correct insertion was then used to construct the final product, as determined by restriction and sequence analysis.

C. The next fragment cloned into the mother plasmid (step 7b) contains a promoterless puromycin gene encoding a sequence with the bovine growth hormone gene poly A signal sequence added at its 3'end after the TGA stop codon. It was a cassette (Fig. 5). The PGK puromycin b poly A plasmid, which is used as a hodient control for puromycin resistance of transformed cells, was digested with HindIII and XhoI. The puromycin b poly A fragment was isolated from the rest of the vector DNA containing the PGK promoter by electrophoresis on a 0.7% agarose gel and purified. The mother plasmid (step 7b) was digested with HindIII and SalI. Hi
The ndIII / XhoI puromycin b poly A cassette was cloned into the mother plasmid Hi.
It was attached to the ndIII and XhoI sites. The ligation was performed according to the manufacturer's recommendations with T4 DNA ligase (New England Biolabs, Bev).
(Ery, MA) with a 3: 1 molar ratio of insert: vector. After ligation, the recombinant plasmid was transformed into a suitable bacterial host (XL1-Blue MRF
, Stratgen, La Jolla, CA). The bacterium was cultivated and plasmid DNA was isolated. To build the final product,
A plasmid with the correct insertion was used as determined by restriction and sequence analysis.

D. The final cloning step involved joining the 5'arm of the construct, which is the 4.616 kb Intron 3 insert from the PCR2.1 vector (step 3). PCR2.1 vector (step 3) was loaded with KpnI and Xho
Digested with I. The 4.616 kb PCR fragment was separated from the vector DNA by agarose gel electrophoresis and purified. Kp of 4.616 kb
The n / Xho I insert was ligated to the KpnI and XhoI digested mother plasmid (step 7c). Ligation was performed in the presence of T4 DNA ligase (New England Biolabs, Beverly, MA) according to the manufacturer's recommendations with an equimolar ratio of insert: vector. After ligation, the recombinant plasmid was transformed into a suitable bacterial host (XL1-Blue MRF ', Strategen,
La Jolla, CA). The bacterium was cultivated and plasmid DNA was isolated by standard molecular biology techniques. The plasmid with the correct insertion, as determined by restriction and sequence analysis, was used as the final product.

E. The final construct was used to transfect porcine embryonic fibroblasts, transgenic porcine fibroblasts, or porcine embryonic stem cells, or porcine primordial germ cells. Cell clones that are resistant to puromycin can be screened by PCR and the integration site determined by methods well known to those of skill in the art. Primers placed in the region of intron 4, which are not incorporated into the final construct,
It can be used in combination with a 5'puromycin gene primer. Only the targeted insert gives a PCR fragment of the appropriate size. All other integration events give rise to negative consequences.

The cell clone with the targeted insert was then cloned using the nuclear transfer technique.
It can be used to generate transgenic animals or, in the case of stem cells, to inject into developing blastocysts and to generate chimeric progeny.

Example 3 (Inactivation of Galα (1,3) galactosyltransferase gene by replacing exon 4 with reverse orientation activity gene) In this example, the Galα (1,3) galactosyltransferase gene was , Was functionally inactivated by using a "collision construct" and inserted an active gene containing splice donor and acceptor sites in place of exons and at least adjacent flanking introns. The inserted gene has a highly active promoter (eg, phosphoglycerate kinase I (PG
K) under the control of the gene promoter), so that transcription of this gene causes termination of transcription of the endogenous gene (Rosario et al., (1996) Na
t. Biotech. 14: 1592-1596). Exon 4 of the Galα (1,3) galactosyltransferase gene encodes the ATG start codon and the N-terminal part of this protein. Therefore, this insert is made to replace exon 4 and a portion of the flanking introns 3 and 4, resulting in a truncated transcript that does not encode a functional enzyme. This example describes targeting introns 3 and 4, but this method is for targeting any intron within the Galα (1,3) galactosyltransferase gene or any other gene of interest. Can be used. A sequence listing of the introns of the Galα (1,3) galactosyltransferase gene (from within intron 3 to the end of intron 8) is provided in FIG.

In this example, the PGK promoter driving the expression of the puromycin resistance gene was inserted along with the bovine growth hormone poly A (b poly A) transcription termination sequence.
This gene uses the intron 3 and 4 sequences for homology flanking the inserted gene by standard homologous recombination techniques to generate Galα (1,3) galactosyltransferase exon 4 and flanking intron 3 And 4
One part of the sequence was replaced. Intron 3, which separates exons 3 and 4, was constructed so that it was greater than 5 kb in length and that at least about 4.6 kb of homologous sequence was present at one end of the gene. Intron 4, which separates exons 4 and 5, is about 6.8 kb in length and is at least about 2.2 k.
The construct was made so that the homologous sequence of b was at the other end of the gene. Positive selection of transfected cells with the construct integrated was achieved by expression of the puromycin resistance gene. As described herein, it is understood that any selectable marker gene that allows targeted cells to survive under appropriate selection conditions can be driven by the strong PGK promoter. In addition, the toxin gene was inserted to eliminate random integration events.

Collision constructs were made using standard molecular biology techniques well known to those skilled in the art.
A 4.616 kb Intron 3 homology fragment and a 2.084 kb Intron 4 homology fragment were generated using PCR and as described in Example 2, steps 1-5 for the displacement targeting constructs above. , PCR2.1 cloning vector. The construction of the collision construct begins with pBS KS +
Incorporating into the vector the 2.084 kb intron 4 homology fragment as the 3'arm of the collision construct, followed by the PGK-puromycin-bovine poly A cassette in the opposite orientation to the coding sequence of the GT gene. The 4.616 kb Intron 3 homology fragment was then cloned as the 5'arm.
This created a targeting construct for recombination on the homologous side. The ricin A toxin gene is also added to the plasmid outside the homologous region, which effectively kills the rate at which random recombination occurs. Ricin A toxin gene
R propagated and cloned based on published sequence (Figure 8). A schematic diagram of the final construct is shown in FIG. In addition, the collision structure was deposited with the ATCC on 28 September 2000 under the deposit number Deposited at. 1. The 2.084 kb 3'arm of the construct (intron 4 homology fragment) contains XhoI-Ec within its multiple cloning site (pBS KS +).
pB modified to include an oRI linker (see Example 2, Step 6)
It was the first fragment that was ligated into the luesscript cloning vector. Ligation of this linker to the vector is described in Example 2, step 7a above,
And 2.084 kb 3'to the EcoRV and SpeI sites of this vector.
Coupling of the arms is described in Example 2, step 7b above. 2. The next step involves ligation of the PGK puromycin bovine poly A cassette into the pBS KS + vector (containing the 2.084 kb 3'arm). This PGK
-Puro-bPA cassette with EcoR immediately 5'to the PGK promoter
Digested with I. This EcoRI overhang was cloned into the Klenow enzyme (Roche
Molecular Biochemicals, Indianapolis,
(IN) using the manufacturer's specifications for smoothing. The PGK-puro-bPA cassette was then released from the vector by digestion with XhoI immediately 3'to the bovine poly A sequence. This smoothed PGK-pu
The ro-bPA-XhoI cassette was subjected to vector D by agarose gel electrophoresis.
Separated from NA and purified. This pBS KS + vector (step 1)
PGK-puro-bPA digested with HpaI and XhoI and blunted
-The XhoI fragment was ligated between the HpaI and XhoI sites of the vector. After ligation, the recombinant plasmid was transformed into a suitable bacterial host (XL1-Blue).
MRF ', Strategene, La Jolla, CA).
The bacterium was cultivated and plasmid DNA was isolated. The plasmid with the correct insert was then used as the final product, as determined by restriction and sequence analysis. 3. The 4.616 kb intron 3 homology fragment was ligated into the pBS KS + mother plasmid (step 2) and the collision construct (collision).
The 5'arm of the construct) is shown. This fragment is PCR2.1
Isolation from the cloning vector, as well as the Kp of this mother plasmid
Ligation to the nI and XhoI sites is described in Example 2, step 7d above. 4. The ricin A toxin gene (FIG. 8) was added to a commercially available mammalian expression vector (eg,
pcDNAI / Amp (Invitrogen)). The insert was then excised with the CMV promoter and SV40 polyA site and cloned into the NotI site of the recombinant plasmid by blunt end ligation after Klenowfilin reaction to both the insert and the vector. 5. After ligation, this recombinant plasmid DNA was used to transform the appropriate bacterial cells (X
LI-blue, Strategene) was transformed. Cultivate this bacterium,
And the plasmid DNA was isolated. This final construct DNA was linearized with KpnI, a unique enzyme site in the plasmid MCS outside the construct sequence. The linearized plasmid could be used to transfect porcine embryonic fibroblasts, transgenic porcine fibroblasts, porcine embryonic stem cells, or porcine primordial germ cells. Puromycin resistant cell clones could be screened by PCR to determine the site of integration by methods well known to those of skill in the art. Primers located in the region of intron 4 that were not incorporated into the final construct could be used in combination with the 5'puromycin gene primer. Only targeted inserts yield PCR fragments of the appropriate size. All other integration events produce negative consequences.

The cell clones with the targeted insert can then be used to produce transgenic animals by using nuclear transfer technology, or, in the case of stem cells, developing blastocysts. And can be used to produce chimeric progeny. Example 4 Isolation of Porcine Genomic DNA Encoding the Galα (1,3) Galactosyltransferase Gene from a λ Phage Clone Library In this example, a porcine genomic library was prepared using molecular biology well known to those skilled in the art. Α (1,3) using specific techniques (eg, Sambrook et al., Supra).
It screened with the cDNA corresponding to the exon 4 of a galactosyl transferase gene. Pig Genome Library (Clontech, Palo Al
to, CA) and were screened with a PCR fragment derived from exon 4 of the porcine Galα (1,3) galactosyltransferase gene. Exon 4 was replaced by the Random Prime Kit (Stratagene,
La Jolla, CA) and labeled with ³² P dCTP according to the manufacturer's instructions. Approximately 4 million phage forming units were screened and a unique clone containing the exon 4 sequence determined by Southern blotting was isolated.
Clones obtained by this procedure contained an insert that was 15-40 kb long. These clones were designated pgGT, λ1, λ2, λ4-1 and λ8-2.
Unique overlapping nucleotide sequences (within Gal3 from intron 3 to intron 8)
Five vectors containing the α (1,3) galactosyl transferase gene have been deposited with the ATCC: (1) 1. within the most 5 ′ end of intron 3 of the 18.275 kb λ-2 phage clone. 6 kb insert, (2) 1
A 6.7 kb HindIII fragment spanning intron 3 to intron 4 of the 8.275 kb lambda-2 phage clone, (3) a 4 kb HindIII behind the 6.7 kb fragment 2 of the 18.275 lambda-2 phage clone.
Fragment, (4) a 6 kb HindIII-SalI fragment in the 3'most part of the 18.275 lambda-2 phage clone, and (5) a 13 kb fragment of the lambda-2 phage clone spanning exons 7-9. These five vectors were deposited with the ATCC on September 29, 2000, each with a registration number Got Subclones of the various inserts were cloned into Intron 3 to Intron 8 provided in Figure 1 using molecular biology techniques well known to those of skill in the art (see, eg, Sambrook et al., Supra). It was used to generate the intron sequences of the present application. These sequences were transformed into porcine Galα (1,3)
It could be used to determine regions of sequence homology in designing targeting constructs for regulation of the galactosyltransferase gene.

Although the compositions and methods provided herein have been described in detail, many modifications and alterations can be made by those of ordinary skill in the art, and such changes and modifications are described herein. It will be appreciated that it can be done without departing from the spirit and scope of the compositions and methods provided herein.

[Brief description of drawings]

FIG. 1 is the nucleotide sequence of the intron of the porcine Galα (1,3) galactosyltransferase gene (from within intron 3 to the end of intron 8).
Indicates. The long syllabary indicates nucleotides within the exon region. Therefore, the numbering of the nucleotide sequences represents the number of bases in the entire porcine Galα (1,3) galactosyltransferase gene in relation to nucleotide position 1 of the insert isolated from the λ-2 phage clone.

FIG. 2 shows a schematic of the gene targeting vector used for inactivation of the porcine Galα (1,3) galactosyltransferase gene (see Example 1). This vector is a promoterless, engineered to contain multiple stop codons (engineered exons) with sequence homology to the 5'region of intron 3 of the Galα (1,3) galactosyltransferase gene. Neomycin phosphotransferase gene, engineered splice acceptor site, 5'region of intron 4 sequence for splicing engineered exon to downstream exon 4, and assists in annealing to porcine Galα (1,3) galactosyltransferase gene For intron 3 is designed to include a sequence having homology to the 3'region of intron 3. The arrow indicates the position of the primer used for PCR.

FIG. 3 shows the nucleotide sequence of a gene targeting vector used to inactivate the porcine Galα (1,3) galactosyltransferase gene (
See Example 1). This vector comprises a sequence having homology to the 5 ′ region of intron 3 of (A.) Galα (1,3) galactosyltransferase gene, (B.) intron 4 splice acceptor sequence, (C.) A promoterless neomycin phosphotransferase gene, (D.) intron 4 splice donor signal sequence, and (E.) porcine Galα (1,3) galactosyltransferase gene engineered to contain a stop codon (engineered exon) 'Intron 3 to help anneal to
Designed to contain an array. All underlined sequences correspond to restriction sites in the primer sequence. Bold type indicates the primer region used for PCR. The usual format indicates a PCR fragment sequence.

FIG. 4 shows the nucleotide sequence for the neomycin phosphotransferase gene (neomycin resistance gene). Bold type indicates the positions of the start and stop codons of the gene. The underlined sequences correspond to the primer sequences.
The capitalized nucleotides are within the coding region of this gene.

FIG. 5 shows the nucleotide sequence of puromycin / bovine growth hormone polyA.
The underlined sequence corresponds to the start codon of the puromycin gene.

FIG. 6 shows a schematic diagram of the gene targeting vector used to inactivate the porcine Galα (1,3) galactosyltransferase gene (see Example 2). This vector contains the Galα (1,3) galactosyltransferase gene 3 ′ intron 3 containing the 3 ′ intron splice acceptor sequence.
Includes a sequence having homology to the sequence, a Kozak consensus sequence, a promoterless puromycin gene engineered to contain the bovine growth hormone polyA sequence (engineered exon), and a 5'intron splice donor sequence. Designed to include sequences with 5'intron 4 sequence homology. Arrow
The position of the primer used for PCR is shown.

FIG. 7 shows the nucleotide sequence of the gene targeting vector schematically shown in FIG. 6 (see Example 2). The underlined sequences correspond to the primer sequences used. Bold type indicates intron regions used for homology. AG and GT at the 3'end of intron 3 and the 5'end of intron 4
Splice consensus sequences are in upper case.

FIG. 8 shows the nucleotide sequence of the Ricin A toxin gene. Capitalized nucleotides are within the coding region of this gene.

FIG. 9 shows a schematic of the collision construct used to inactivate the porcine Galα (1,3) galactosyltransferase gene (see Example 3). This vector contains Galα (1,3 containing a 3'intron splice acceptor sequence.
) A sequence having homology to the galactosyltransferase gene 3'intron 3 sequence, a reverse orientation puromycin gene engineered to contain the bovine growth hormone poly A sequence under the control of the phosphoglycerate kinase (PGK) promoter, And a 5'intron 4 containing a 5'intron splice donor sequence and a ricin A toxin gene under the control of the cytomegalovirus (CMV) promoter
It has sequence homology and is designed to include sequences containing the SV40 polyA sequence located outside the region of homology. The arrow indicates the position of the primer used for PCR.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C N, CR, CU, CZ, DE, DK, DM, EE, ES , FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, K R, KZ, LC, LK, LR, LS, LT, LU, LV , MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, S I, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ramsundahr, Hagdis Jae             I.             United States New York 13346,               Hamilton, Madison Street               46 F-term (reference) 4B024 AA01 AA20 CA04 DA02 DA06                       EA04 FA10 FA18 GA11 HA01                 4B065 AA26X AA90X AA99Y AB01                       AC20 BA02 CA44 CA46                 4C081 AA01 AA12 AA14 AB01 AB11                       AB31 BA12 CD34

Claims (71)

[Claims] 1. A method of modulating the expression of a eukaryotic gene in a cell, said method comprising the step of transfecting said cell with a nucleic acid construct, said construct comprising:
A first construct sequence homologous to said gene sequence, a sequence encoding a selectable marker, and a second construct sequence homologous to a second gene sequence, said first and second gene sequences comprising said eukaryotic Independently comprising at least a portion of one or more intron regions of a biological gene, and incorporating the selectable marker into the eukaryotic gene, the expression of the selectable marker being the expression of the selectable marker in the cell. A method which results in the regulation of expression of a nucleo gene. 2. The method of claim 1, wherein the first construct sequence and the second construct sequence are each homologous to at least a portion of an intron region of the gene. 3. The method of claim 1, wherein the sequence encoding the selectable marker is integrated into the eukaryotic gene by homologous recombination and the first construct sequence is the first construct sequence. The method, wherein the selectable marker is inserted into the gene by recombination with a gene sequence and the second construct sequence with the second gene sequence. 4. The method of claim 1, further comprising screening the cells for expression of the selectable marker. 5. The method of claim 2, wherein the first construct sequence and the second construct sequence are homologous to different regions within the same intron of the gene. 6. The method of claim 2, wherein the first construct sequence and the second construct sequence are homologous to regions of different introns. 7. The method according to claim 1, wherein the selectable marker gene is an antibiotic resistance gene. 8. The method of claim 1, wherein the selectable marker-encoding sequence, when expressed, is ampicillin resistance, kanamycin resistance, geneticin resistance, neomycin resistance, puromycin resistance, hygromycin b. A nucleotide sequence conferring resistance, thymidine kinase activity, tryptophan synthetase activity, adenine phosphoribosyl transferase activity, dihydrofolate reductase activity, and a phenotype selected from the group consisting of histidinol dehydrogenase, anthocyanin, β-glucuronidase and luciferase. ,Method. 9. The method of claim 8, wherein the selectable marker coding sequence confers neomycin resistance or puromycin resistance. 10. The method of claim 1, wherein the eukaryotic gene is
B7.3, P-selectin, E-selectin, ICAM-1, ICAM-2, V
CAM-1, CD28, CD80, CD86, CD154, major histocompatibility complex class I, β-2 macroglobulin, invariant chain, caspase-
1. A method selected from the group consisting of genes encoding 1, caspase-3, and Galα (1,3) galactosyltransferase. 11. The method of claim 10, wherein the eukaryotic gene encodes Galα (1,3) galactosyltransferase. 12. The method of claim 11, wherein the Galα (1,3
) The method wherein the galactosyltransferase gene is a porcine gene. 13. The method of claim 12, wherein the first construct sequence and the second construct sequence are intron 3, intron 4, intron of the porcine Galα (1,3) galactosyltransferase gene. 5, independently selected from the homologous region of the intron selected from the group consisting of 5, intron 6, intron 7, intron 8 and intron 9. 14. The method of claim 13, wherein the intron 4 is
The method having the nucleotide sequence of nucleotides 4938-11716 of FIG. 15. The method of claim 13, wherein the intron 5 is
The method having the nucleotide sequence of nucleotides 11753-13748 of Figure 1. 16. The method of claim 13, wherein the intron 6 is
A method having the nucleotide sequence of nucleotides 13810-14358 of FIG. 17. The method of claim 13, wherein the intron 7 is
The method having the nucleotide sequence of nucleotides 14463-21627 of FIG. 18. The method of claim 13, wherein the intron 8 is
The method having the nucleotide sequence of nucleotides 21766 to 27048 of Figure 1. 19. The method of claim 13, wherein the first construct sequence and the second construct sequence are homologous to different regions within the same intron of the eukaryotic gene. 20. The method of claim 19, wherein the intron is intron 3 of the porcine Galα (1,3) galactosyltransferase gene. 21. The method of claim 13, wherein the first construct sequence and the second construct sequence are homologous to different introns of the porcine Galα (1,3) galactosyltransferase gene. . 22. The method of claim 21, wherein the first construct sequence is upstream of the second construct sequence. 23. The method of claim 21, wherein the intron region of the first construct is homologous to the intron 3 region and the intron region of the second construct is porcine Galα (1, 3) A method which is homologous to the intron 4 region of galactosyltransferase. 24. The method of claim 2, wherein the sequence encoding the selectable marker is a promoterless gene. 25. The method of claim 2, wherein the selectable marker coding sequence further comprises a promoter. 26. The method of claim 25, wherein the promoter is
The method, which is a phosphoglycerate kinase (PGK) promoter. 27. The method of claim 2, wherein the sequence encoding the selectable marker is transcribed in an orientation opposite to that of the eukaryotic gene. 28. The method of claim 27, wherein the sequence encoding the selectable marker further comprises a promoter sequence. 29. The method of claim 1, wherein the cells are from the group consisting of fibroblasts, epithelial cells, endothelial cells, transgenic embryonic fibroblasts, embryonic stem cells, and primordial germ cells. The method chosen. 30. The method of claim 2, wherein the cells are pig cells. 31. The method of claim 2, wherein the construct further comprises an AG dinucleotide splice acceptor site. 32. The method of claim 2, wherein the construct further comprises a GT dinucleotide splice donor site. 33. A nucleic acid construct comprising a first construct sequence homologous to a first gene, a sequence encoding a selectable marker, and a second construct sequence homologous to a second gene sequence, said first construct sequence comprising: And a second gene sequence independently comprising at least a portion of one or more intron regions of a eukaryotic gene. 34. The nucleic acid construct of claim 33, further comprising an AG dinucleotide splice acceptor site. 35. The nucleic acid construct of claim 33, further comprising a GT dinucleotide splice donor site. 36. The nucleic acid construct of claim 33, further comprising a Kozak consensus sequence. 37. The nucleic acid construct of claim 33, wherein the sequence encoding the selectable marker, when expressed, is ampicillin resistance, kanamycin resistance, geneticin resistance, neomycin resistance, puromycin resistance, hygromycin. a nucleotide sequence conferring b resistance, thymidine kinase activity, tryptophan synthetase activity, adenine phosphoribosyl transferase activity, dihydrofolate reductase activity, and a phenotype selected from the group consisting of histidinol dehydrogenase, anthocyanins, β-glucuronidase and luciferase. A nucleic acid construct. 38. The nucleic acid construct of claim 37, wherein the sequence encoding the selectable marker confers puromycin resistance or neomycin resistance.
Nucleic acid construct. 39. The nucleic acid construct according to claim 33, wherein the eukaryotic gene is B7.3, P-selectin, E-selectin, ICAM-1, ICAM.
-2, VCAM-1, CD28, CD80, CD86, CD154, major histocompatibility complex class I, β-2 macroglobulin, invariant chain, caspase-1, caspase-3, and Galα (1,3). A nucleic acid construct selected from a gene encoding a galactosyltransferase. 40. The nucleic acid construct of claim 39, wherein the eukaryotic gene is porcine Galα (1,3) galactosyltransferase. 41. The nucleic acid construct of claim 40, wherein the first construct sequence and the second construct sequence are intron 3, intron 4, of the porcine Galα (1,3) galactosyltransferase gene. A nucleic acid construct independently selected from a homologous region of an intron selected from the group consisting of intron 5, intron 6, intron 7, intron 8 and intron 9. 42. The nucleic acid construct of claim 41, wherein intron 4 has the nucleotide sequence of nucleotides 4938 to 11716 of FIG. 1 and intron 5 has the nucleotide sequence of nucleotides 11753 to 13748 of FIG. 1, intron 6 has the nucleotide sequence of nucleotides 13810-14358 of FIG. 1, intron 7 has the nucleotide sequence of nucleotides 14463-21627 of FIG. 1, and intron 8 is of nucleotide 2176 of FIG.
A nucleic acid construct having a nucleotide sequence from 6 to 27048. 43. The method of claim 41, wherein the first construct sequence and the second construct sequence are homologous to different regions within the same intron of the eukaryotic gene. . 44. The nucleic acid construct of claim 43, wherein the intron is intron 3 of the porcine Galα (1,3) galactosyltransferase gene. 45. The nucleic acid construct of claim 41, wherein the first construct sequence and the second construct sequence are homologous to different introns of the porcine Galα (1,3) galactosyltransferase gene. Nucleic acid construct. 46. The nucleic acid construct of claim 45, wherein the first construct sequence is homologous to the intron 3 region of porcine Galα (1,3) galactosyltransferase and the second construct sequence. But pig Galα (1,3
) A nucleic acid construct that is homologous to the intron 4 region of galactosyl transferase. 47. Transfected with the nucleic acid construct of claim 33,
cell. 48. Transfected with the nucleic acid construct of claim 41,
cell. 49. transfected with a nucleic acid construct according to claim 44,
cell. 50. Transfected with the nucleic acid construct of claim 46,
cell. 51. A bacterial cell transformed with the nucleic acid construct of claim 33. 52. A bacterial cell transformed with the nucleic acid construct of claim 41. 53. A bacterial cell transformed with the nucleic acid construct of claim 44. 54. A bacterial cell transformed with the nucleic acid construct of claim 46. 55. Gal with nucleotides 4938-11716 of FIG.
Nucleotide sequence of intron 4 of the α (1,3) galactosyltransferase gene. 56. Ga having nucleotides 11753-13748 of FIG.
Nucleotide sequence of intron 5 of the lα (1,3) galactosyltransferase gene. 57. Ga having nucleotides 13810-14358 of Figure 1
Nucleotide sequence of intron 6 of the lα (1,3) galactosyltransferase gene. 58. Ga having nucleotides 14463-21627 of FIG.
Nucleotide sequence of intron 7 of the lα (1,3) galactosyltransferase gene. 59. Ga having nucleotides 21766-27048 of FIG.
Nucleotide sequence of intron 8 of the lα (1,3) galactosyltransferase gene. 60. A λ phage clone derived from a porcine genomic library containing at least part of the Galα (1,3) galactosyltransferase gene, wherein the λ phage clone is pgGT, λ1, λ2, λ4-1 and λ8.
Λ phage clone selected from the group consisting of -2. 61. A method of producing a transgenic mammal, the method comprising the step of transfecting a nuclear donor cell with the nucleic acid construct of claim 33, the transfected cell comprising nucleic acid of said nucleic acid construct. A method of introducing the selected cells into an embryo, fertilizing the embryo into a suitable host mammal, and producing progeny from the fertilized host mammal. 62. A method of producing a transgenic mammal, the method comprising the step of transfecting a nuclear donor cell with the nucleic acid construct of claim 44, the transfected cell comprising nucleic acid of said nucleic acid construct. A method of introducing the selected cells into an embryo, fertilizing the embryo into a suitable host mammal, and producing progeny from the fertilized host mammal. 63. A method of producing a transgenic mammal, the method comprising transfecting a nuclear donor cell with the nucleic acid construct of claim 46, the transfected cell comprising the nucleic acid of the construct. A method comprising the steps of selecting, introducing the selected cells into an embryo, fertilizing the embryo into a suitable host mammal, and producing progeny from the fertilized host mammal. 64. A transgenic mammal produced according to the method of claim 61. 65. A transgenic mammal produced according to the method of claim 62. 66. A transgenic mammal produced according to the method of claim 63. 67. A method of reducing transplant rejection, the method comprising the method of claim 32.
Transfecting a nuclear donor cell with the nucleic acid construct of claim 1, selecting a transfected cell containing the nucleic acid of the construct, introducing the selected cell into an embryo, introducing the embryo into a suitable host mammal. Conception, producing progeny from the conception host mammal, collecting cells, tissues, or organs from the progeny, and transplanting the collected cells, tissues, or organs into a patient in need thereof A method comprising the step of: 68. A method of reducing transplant rejection, the method comprising the method of claim 44.
Transfecting a nuclear donor cell with the nucleic acid construct of claim 1, selecting a transfected cell containing the nucleic acid of the construct, introducing the selected cell into an embryo, introducing the embryo into a suitable host mammal. Conception, producing progeny from the conception host mammal, collecting cells, tissues, or organs from the progeny, and transplanting the collected cells, tissues, or organs into a patient in need thereof A method comprising the step of: 69. A method of reducing transplant rejection, the method comprising:
Transfecting a nuclear donor cell with the nucleic acid construct of claim 1, selecting a transfected cell containing the nucleic acid of the construct, introducing the selected cell into an embryo, introducing the embryo into a suitable host mammal. Conception, producing progeny from the conception host mammal, collecting cells, tissues, or organs from the progeny, and transplanting the collected cells, tissues, or organs into a patient in need thereof A method comprising the step of: 70. The nucleic acid construct of claim 43, further comprising a nucleic acid sequence encoding a gene that is toxic to the eukaryotic cell. 71. The nucleic acid construct according to claim 70, wherein the gene toxic to the eukaryotic cell is the ricin A toxin gene.