CN111296364B

CN111296364B - Gene modification method for mouse animal model and application thereof

Info

Publication number: CN111296364B
Application number: CN201911027061.6A
Authority: CN
Inventors: 徐俊; 程露; 刘业恒; 易佳炜; 周雪峰
Original assignee: SHANGHAI RAAS BLOOD PRODUCTS CO Ltd
Current assignee: SHANGHAI RAAS BLOOD PRODUCTS CO Ltd
Priority date: 2019-10-27
Filing date: 2019-10-27
Publication date: 2022-06-24
Anticipated expiration: 2039-10-27
Also published as: CN111296364A

Abstract

The invention relates to a gene modification method of a genetically modified mouse animal model and application of the mouse model in screening targeted drugs. The invention generates a humanized protein C knock-in mouse by using a human protein C gene expression cassette to knockout and replace a mouse protein C gene in a targeted manner. The mice are reproductive and can be crossed with other mouse disease models (e.g., mice lacking clotting factor VIII or IX) to produce humanized protein C mouse disease models (e.g., a humanized protein C-depleted factor VIII mouse model or a humanized protein C-depleted factor IX mouse model). These mouse models can be used to study the function of human protein C and human activated protein C (apc) in vivo. The mouse models are the first animal models of therapeutic candidate drugs for testing the target human protein C or APC in vivo, and have high economic value and scientific research value.

Description

Gene modification method of mouse animal model and application thereof

Technical Field

The invention relates to a gene modification method of a genetically modified mouse animal model and application of the animal model in screening targeted drugs. The mouse models are the first animal models of therapeutic candidate drugs for testing the target human protein C or APC in vivo, and have high economic value and scientific research value.

Background

The invention relates to a gene modification method of a mouse animal model, which replaces a nucleotide sequence which codes a certain endogenous protein in the original mouse genome with a human nucleotide sequence which codes homology. And provides vectors, cells and methods for producing such mouse animal models. The invention also provides a method for detecting the expression and/or functional activity of the human protein in a mouse body, thereby laying a foundation for the research of drug effect.

The human protein C pathway plays a key role in the regulation of coagulation and inflammatory responses. The activation reaction of human protein C is generated by thrombin binding to thrombomodulin on endothelial cells. Protein C is a zymogen that is converted to activated protein C (apc) by the thrombomodulin/thrombin complex.

Activated protein c (apc) is a serine protease. APC cleavage of activated factor V and activated factor VIII negatively regulates thrombin formation, which is critical for maintaining a balance of thrombosis and hemostasis in vivo. In other words, APC inhibits the main coagulation driving force. Thus, APC has an important anticoagulant function, the main goal of which is activated factor V and activated factor VIII. Human APCs can also enhance the fibrinolytic response through the formation of complexes with plasminogen activator inhibitors.

Protein C/APC has become an attractive therapeutic target in view of its important physiological activity. For example, protein C/APC, an important role in coagulation regulation as an anticoagulant, has been used as a therapeutic target in the context of hemophilia (poldedijk et al, curr. opin. hematol.,2017,24: 446-. Hemophilia is a serious bleeding disorder, mainly caused by deficiencies or deletions of factor VIII or factor IX. Treatment of hemophilia typically involves prophylactic or on-demand injection of missing or deficient factors. Such treatments are expensive and patients receiving such treatments also produce inhibitory antibodies to the injected factor, requiring the use of "bypass" agents. Suitable replacement therapies are urgently needed by hemophiliacs.

In addition to its anticoagulant function, APC also has cytoprotective activity, including anti-inflammatory and anti-apoptotic activity, as well as protection of endothelial barrier function (Krisinger et al, FEBS Journal,2009,276: 6586-. Thus, APC is also a therapeutic target in the field of inflammatory and/or apoptotic diseases, such as sepsis.

Although protein C and APC have several important functions, it is difficult to study protein C and APC comprehensively in vivo since mice lacking protein C die as soon as they are born. (Jalbert et al, J.Clin. invest.,1998,102(8): 1481-8).

Human protein C (or human APC) is a potential therapeutic target for humans, and its amino acid sequence differs significantly from that of mice. The homology of amino acid sequences of mouse protein C and human protein C is only 69%. The significant difference in sequence (structure) between mouse protein C and human protein C means that it is highly undesirable to use mouse protein C to study the potential therapeutic effects of human protein C. Furthermore, ex vivo experiments between species showed that human protein C does not function effectively in mouse plasma (Krisinger et al, FEBS Journal,2009,276: 6586-.

Transgenic non-human animals, such as mice, that replace endogenous non-human protein C with sequences that express human protein C, if produced and normally propagated, are very useful models for in vivo study of human protein C, for example for testing potential therapeutic drugs that target human protein C. However, since protein C-deficient mice (i.e., protein C-free mice) die at birth and it has been reported that human protein C does not function effectively in mouse plasma, such a genetically modified animal, such as a mouse, is not possible.

However, surprisingly, the present inventors have been able to make such genetically engineered mice. Such mice can survive healthily and have bleeding characteristics consistent with those of control mice expressing mouse protein C.

Disclosure of Invention

In a first aspect, the present invention provides a method of genetically modifying a genetically modified non-human animal model, wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced by a functional fragment encoding human protein C or a functional variant encoding human protein C.

In some embodiments, the non-human animal is a non-human mammal. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse or rat, preferably a laboratory mouse or rat.

A preferred non-human animal of the invention is a mouse. The strain of this mouse is the laboratory mouse C57 BL/6.

The non-human animal of the invention is genetically modified. This means that these animals are not naturally occurring animals, i.e. not natural or wild-type animals. The genetic engineering is by technical means as described elsewhere herein. According to the invention, the genetic modification comprises replacing at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of said non-human animal with a nucleotide sequence encoding a functional fragment or a functional variant of human protein C.

The non-human animals of the invention express or are capable of expressing a functional fragment or functional variant of human protein C.

An endogenous nucleotide sequence encoding protein C refers to a nucleotide sequence encoding protein C for a non-human animal, i.e., a wild-type (or native or endogenous).

The coding sequence (CDS) of endogenous protein C is a nucleotide sequence that encodes (or corresponds to) an amino acid sequence in a protein C protein. CDS starts with a start codon (usually ATG) and ends with a stop codon (e.g. TAG). However, as is known to those skilled in the art, in the context of endogenous animal genes (i.e. in the genomic context), the coding sequence of protein C (exons) is usually interrupted by introns, which means that the CDS itself exists as a discontinuous nucleotide sequence in an endogenous gene.

Thus, in a preferred embodiment of the invention, the endogenous nucleotide sequence encoding protein C that has been replaced is a nucleotide sequence that begins, in whole or in part, with a start codon and ends with a stop codon, including other exon and intron sequences located between the start codon and the stop codon. In a preferred embodiment, the entire (i.e., all) nucleotide sequence from the start codon to the stop codon has been replaced, including other exon and intron sequences between the start codon and the stop codon.

As noted above, mice are particularly preferred non-human animals of the invention. Mouse protein C refers to protein C to wild-type (or native) mice. The amino acid sequence of mouse protein C is shown herein as SEQ ID NO: 12. the nucleotide coding sequence (CDS) of mouse protein C is shown herein as SEQ ID NO: 10. mouse protein C (Proc) gene (NCBI reference sequence: NM-001042767.3) is located on mouse chromosome 18. 9 exons have been identified, with the start codon (ATG) located in exon 2 and the stop codon (TAG) located in exon 0.

Thus, in a preferred embodiment of the invention, the endogenous nucleotide sequence encoding mouse protein C that has been replaced is all or part of the nucleotide sequence from the start codon (ATG) in exon 2 to the stop codon in exon 9 of the mouse protein C gene, including other exon and intron sequences between the start codon and the stop codon. In a preferred embodiment, the entire (i.e., all) nucleotide sequence from the start codon (ATG) of exon 2 of the mouse protein C gene to the stop codon of exon 9 of the mouse protein C gene, including other exon and intron sequences between the start and stop codons, is replaced.

As a result of the replacement of at least one copy of the endogenous nucleotide sequence encoding protein C, at least one allele of the protein C gene in the non-human animal does not encode wild-type protein C, as described in the present invention. Thus, in a non-human animal, at least one allele of protein C does not express (and is not capable of expressing) the animal's wild-type (or native) mRNA encoding protein C.

In the mouse case, at least one allele of the protein C gene in the genetically modified mouse of the invention does not encode the wild-type (or native or endogenous) protein C of the mouse. Thus, at least one allele of the protein C gene in the mouse does not express an mRNA molecule encoding protein C from the wild-type (or native) mouse, the mRNA molecule encoding sequence is set forth in SEQ ID NO:12 (the corresponding nucleotide sequence is shown in SEQ ID NO: 10).

Human protein C refers to human wild-type (or native or endogenous) protein C. The amino acid sequence of human protein C is shown herein as SEQ ID NO: 11. the nucleotide coding sequence (CDS) of human protein C is shown herein as SEQ ID NO: 8. in the context of the present invention, a nucleotide sequence encoding human protein C, i.e. a nucleotide sequence encoding a functional fragment of human protein C or encoding a functional mutant of human protein C, is considered to be a heterologous (or foreign) nucleotide sequence.

Preferably, at least one copy of the endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced by a nucleotide sequence encoding human protein C (i.e., full-length or wild-type human protein C). Thus, preferably, at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced by a nucleotide sequence encoding human protein C, wherein the amino acids (or the composition thereof) of human protein C are as set forth in SEQ ID NO:11 sequence. A typical nucleotide sequence encoding human protein C (SEQ ID NO: 11) is set forth in SEQ ID NO:8, in the list. The nucleotide sequence encoding human protein C may be selected (or composed) of the nucleotide sequence set forth in SEQ ID NO:8, such as SEQ ID NO:8, codon degenerate version.

Generally, the nucleotide sequence encoding human protein C is the coding sequence (CDS) of the human protein C gene, i.e. without inclusion of the intron and the non-coding UTR regions of the human protein C gene.

In an alternative embodiment, the nucleotide sequence encoding human protein C may additionally comprise an intron sequence of the human protein C gene. In some embodiments, a human 5'UTR and/or human 3' UTR nucleotide sequence may be added. The human protein C (PROC) gene (NCBI reference sequence: NM-000312.3) is located on human chromosome 2. 9 exons have been identified, an initiation codon (ATG) in exon 2, and a stop codon (TAG) in exon 9.

In some alternative embodiments, at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced with a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C (i.e., in place of full-length wild-type human protein C).

A "functional" fragment of human protein C or a "functional" mutant of human protein C refers to a fragment or mutant that exhibits (or maintains) at least one functional activity of full-length wild-type human protein C, e.g., a mutant. Exhibits 10%, 25%, 75%, 90% or 100% of the level of at least one functional activity of the full-length wild-type human C protein. For example, anticoagulant activity and/or cytoprotective activity (e.g., anti-inflammatory activity and/or anti-apoptotic activity), as described elsewhere herein.

In some embodiments, fragments of human protein C may be at least 100, 150, 200, 50, 300, 350, 400, or 450 contiguous amino acids in length. In some embodiments, the fragment may be up to 460 contiguous amino acids in length (e.g., 460, 200, 300, or 400 contiguous amino acids in length).

In some embodiments, the fragment of human protein C is a naturally occurring fragment of human protein C, such as activated protein C (apc).

In some embodiments, the mutant of human protein C is a protein having an amino acid sequence that is substantially homologous to the amino acid sequence of full-length wild-type human protein C (or a fragment thereof).

The term "substantially homologous" as used herein in connection with an amino acid or nucleic acid sequence includes having at least 75%, 80%, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the disclosed amino acid or nucleic acid sequence. Thus, substantially homologous sequences of the invention include single or multiple base or amino acid changes (additions, substitutions, insertions or deletions). At the amino acid level, preferred substantially homologous sequences contain up to 10 or up to 5, for example only 1, 2, 3, 4 or 5, preferably 1, 2 or 3, more preferably 1 or 2, altered amino acids. The changes may be conservative or non-conservative amino acids. Preferably, the alteration is a conservative amino acid substitution.

As used herein, a "conservative amino acid substitution" is an amino acid in which an amino acid residue is substituted with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including primary side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

Methods for performing manipulation of amino acids and protein domains (e.g., to generate substantially homologous sequences) are well known to those skilled in the art. Such manipulations can conveniently be carried out at the nucleic acid level by genetic engineering, in which nucleic acid molecules encoding the appropriate proteins are modified so that the amino acid sequences of the resulting expressed proteins are in turn modified in an appropriate manner.

Mutants of human protein C (or fragments thereof) also include modified forms of human protein C (or fragments thereof), such as human protein C (or fragments thereof) that comprise one or more additional amino acids at one or both termini (e.g., comprising one or more N-terminal and/or C-terminal fusion moieties or fusion tags or epitope tags). Thus, the mutant may comprise a fusion protein comprising the amino acid sequence of human protein C (or a fragment of human protein C).

The degree of homology between the sequences can be assessed by any convenient method. However, in order to determine the degree of homology between sequences, computer programs are useful which perform multiple alignments of sequences, for example Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res, 22: 4673-. The Clustal W algorithm may be used with the BLOSUM 62 scoring matrix, if desired (Henikoff and Henikoff,Proc. Natl. Acad. Sci. USA89: 10915-. Other methods that can be used to align sequences are the Needleman and Wunsch alignment methods (Needleman and Wunsch,J. Mol. Biol.48:443, 1970), by Smith and Waterman (Smith and Waterman,Adv. Appl. Math.2:482, 1981) was revised so that the highest order match was obtained between the two sequences and the number of identical amino acids between the two sequences was determined. Between two amino acid sequencesOther methods of similar percentages also include the methods described by Carillo and Lipton (Carillo and Lipton,SIAM J. Applied Math1073, 1988) and lesk editors, "biological calculations, new york oxford university press, 1988: methods described in molecular biology were calculated in the informatics and genomics programs.

Alignment of sequences is typically performed using computer programs, such as ALIGN (Myers and Miller,CABIOS, 4:11-17, 1988），FASTA（Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology183:63-98, 1990) and gapped BLAST (Altschul)et al., Nucleic Acids Res25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies,Nucleic Acids Res., 12:387, 1984). In addition, the Dali server of the european bioinformatics institute may also provide structural alignments of protein sequences (Holm,Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998）。

by providing a reference point, the sequences of the present invention can have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence similarity, etc. data determined under the default parameters of the ALIGN program (e.g., available from GENESTREAM web servers of IGH, Montelier, France).

According to the invention, at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced by a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C.

Typically, the non-human animals of the invention are diploid, which means that their somatic chromosomes have two homologous copies (or homologs). In a normal (unmodified or wild-type) diploid animal, an endogenous protein C gene is found on each of the two chromosomes of the relevant chromosome pair. For example, in normal (wild-type) mice, the protein C gene is encoded on chromosome 18, so in normal mouse somatic cells, there will be two copies of the mouse protein C gene, one on each chromosome 18.

In some embodiments, one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced with a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C. Thus, in some embodiments of the invention, the human protein C genetically engineered non-human animal is heterozygous. Such a heterozygote may be referred to as hproC +/-.

In a preferred embodiment, two (i.e., all) copies of the endogenous nucleotide sequence encoding protein C in the genome of the non-human animal have been replaced with a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C. Thus, in a preferred embodiment of the invention, the genetically modified non-human animal is homozygous for its human protein C allele. Such homozygotes may be referred to as hproC + +. For the avoidance of doubt, such homozygous animals do not comprise in their genome a nucleotide sequence encoding protein C of the wild type (or native) of the animal.

In a particularly preferred embodiment, the invention provides a genetically engineered mouse in which both copies of an endogenous nucleotide sequence encoding protein C in the mouse genome are replaced with a nucleotide sequence encoding human protein C.

Reference herein to a "human protein C allele" or "human protein C allele of the invention" is an abbreviation for the allele of the genome of the non-human animal in which the endogenous nucleotide sequence encoding protein C is replaced by a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant encoding human protein C.

Reference herein to "at least one copy" or "at least one allele" (or the like) generally means one or two copies or one or two alleles, preferably two copies or two alleles.

The nucleotide sequence encoding protein C discussed herein is a nucleotide sequence in a nucleic acid molecule. Thus, they may be regarded as nucleic acid molecules, typically DNA molecules, comprising (or consisting of) said nucleotide sequence.

Reference herein to the "genome" of a genetically modified non-human animal generally refers to the genome of a somatic (or diploid) cell of the non-human animal of the invention. Of course, the human protein C alleles of the invention may also be present in the genome (haploid genome) of some or all (or substantially all) germ cells (eggs or sperm) of genetically modified animals of the invention.

In the context of the present invention, a "substitution by … …" means that the endogenous (or wild-type or native) protein C allele in the genome of the non-human animal has been modified to not comprise (encode) the endogenous protein C encoded by the endogenous nucleotide sequence, and that the allele is substituted with a nucleotide sequence comprising a nucleotide sequence encoding human protein C or a functional fragment or mutant thereof. Thus, the endogenous nucleotide sequence encoding endogenous protein C has been deleted and in its place a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof is present. Preferably, both protein C alleles have been so modified.

In a preferred embodiment, the start codon (e.g. ATG) of the nucleotide sequence encoding human protein C or a functional fragment or mutant thereof is located (or located or inserted) in a protein C gene in the non-human genome, which position corresponds to the start codon of the nucleotide sequence encoding endogenous protein C. And a stop codon (e.g., TAG) of the nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof is located in the genome of the non-human animal in a position corresponding to the stop codon of the nucleotide sequence encoding endogenous protein C in the protein C gene.

In some preferred genetically modified mice of the invention, the start codon (e.g. ATG) of the nucleotide sequence encoding human protein C or a functional fragment or mutant thereof is located (or located or inserted) at a position corresponding to the start codon (ATG) of exon 2 of the mouse protein C gene, and the stop codon (e.g. TAG) of the nucleotide sequence encoding human protein C or a functional fragment or mutant thereof is located (or located or inserted) at a position corresponding to the stop codon (TAG) of exon 9 of the mouse protein C gene.

The start codon (ATG) of exon 2 of the mouse protein C gene (endogenous or wild-type mouse protein C gene) is located immediately adjacent to the 5'-UTR (untranslated region) of exon 2, i.e., immediately 3' or immediately downstream. The stop codon (TAG) of exon 9 of the mouse Protein C gene (endogenous or wild-type mouse Protein C gene) is located in front of, i.e.immediately 5 'or upstream of, the 3' -UTR (untranslated region) of exon 9.

Thus, in some preferred embodiments of the genetically modified mice of the invention, the start codon (e.g. ATG) of the nucleotide sequence encoding human protein C or a functional fragment or mutant thereof is located (or located or inserted) after, i.e. immediately 3 'or immediately downstream of, the 5' -UTR (untranslated region) portion of exon 2 in the mouse genome, and the stop codon (e.g. TAG) of the nucleotide sequence encoding human protein C or a functional fragment or mutant thereof is located (or located or inserted) before, i.e. immediately 5 'or upstream of, the 3' -UTR (untranslated region) portion of exon 9 in the mouse genome. The 5' -UTR (untranslated region) portion of exon 2 of the mouse protein C gene is shown herein as SEQ ID NO: 5. the nucleotide sequence of the 3' -UTR (untranslated region) portion of exon 9 of the mouse protein C gene is represented herein as SEQ ID NO: 6.

in addition, the endogenous nucleotide sequence encoding endogenous protein C in the genome of the non-human animal (e.g., mouse) has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or mutant thereof. Preferably, both protein C alleles are replaced simultaneously.

In addition, the non-human animal (e.g., mouse) of the invention comprises a targeted insertion of a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof into at least one copy of the protein C gene. Due to the targeted insertion of the gene, at least one copy of the protein C gene comprises a nucleotide sequence capable of expressing a gene encoding human protein C or a functional fragment or a functional mutant thereof and does not comprise an endogenous (wild-type) nucleotide sequence encoding endogenous protein C. Preferably, two protein C alleles inserted into the genome of the non-human animal are targeted.

In addition, the non-human animal (e.g., mouse) of the present invention is characterized in that the nucleotide sequence encoding the endogenous protein C in the genome of the non-human animal is targeted-replaced with a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof. Preferably, the targeted replacement of both protein C alleles in the genome of the non-human animal.

The modified allele is referred to as a "knock-in" allele (or KI allele), for example at least one protein C allele in the genome of a non-human animal into which a nucleotide sequence encoding human protein C (or encoding a functional fragment or mutant thereof) is to be "knocked in". Such "knockin" results in the removal of the endogenous nucleotide sequence encoding endogenous protein C. Thus, the modified (knock-in) allele has a sequence that encodes a nucleotide endogenous to the endogenous protein that is knocked out. Thus, the nucleotide sequence encoding endogenous protein C is replaced by a nucleotide sequence encoding human protein C (or a functional fragment or a functional mutant thereof). The "knock-in" allele according to the invention is preferably a constitutive knock-in allele.

It is well known to the skilled person how to identify whether at least one copy (e.g. one copy or two copies) of an endogenous nucleotide sequence encoding protein C in the genome of a non-human animal has been replaced by a nucleotide sequence encoding human protein C (or a functional fragment or mutant). For example, genotyping, such as PCR-based genotyping methods and/or Southern blotting, may be used. Methods of identification are described in the examples section herein.

In some embodiments, the non-human animal of the invention is made using a vector of the invention.

Thus, in some embodiments, a non-human animal of the invention comprises in its genome at least one allele of a protein C gene, comprising (i) a nucleotide sequence of a 5 'untranslated region (UTR) of the protein C gene of the non-human animal, (ii) a nucleotide sequence encoding human protein C, or a functional fragment or mutant thereof, and (iii) a nucleotide sequence of a 3' untranslated region (UTR) of the protein C gene of the non-human animal. (i) The fragments (ii) and (iii) are typically distributed in the 5 'to 3' direction next to each other (i.e. (i) followed by (ii) and then by (iii)). In some embodiments, the marker for positive selection may be located at the 5 'end (corresponding to the 5' -UTR) or at the 3 'end (corresponding to the 3' UTR). Markers for positive selection may be flanked by site-specific recombination sites (e.g., LoxP sites). In some embodiments, the marker used for positive selection may have been removed (e.g., by a site-specific recombinase, such as Cre recombinase). In embodiments where the marker for positive selection is removed by a site-specific recombinase (e.g., Cre), the nucleotide sequence of a single site-specific recombination site typically remains in the allele.

In some embodiments, the non-human animal of the invention is a mouse comprising in its genome at least one protein C allele comprising (i) a nucleotide sequence of the 5 'untranslated region (UTR) of exon 2 of the mouse protein C gene (e.g., SEQ ID NO: 5), (ii) a nucleotide sequence encoding human protein C, and (iii) a nucleotide sequence of the 3' untranslated region (UTR) of exon 9 of the mouse protein C gene (e.g., SEQ ID NO: 6). (i) The fragments (ii) and (iii) are generally distributed next to each other in the 5 'to 3' direction in that order. In some embodiments, the marker for positive selection (e.g., Neor) may be located at the 5 'end (corresponding to the 5' -UTR) or at the 3 'end (corresponding to the 3' UTR), with a preference for 3 '(corresponding to the 3' UTR). Markers for positive selection may be flanked by site-specific recombination sites (e.g., LoxP sites). Preferably, such alleles are substantially as shown in figure 1C herein. In some embodiments, the non-human animal of the invention is a mouse comprising in its genome at least one allele of a protein C gene, the allele comprising the amino acid sequence of SEQ ID NO: 9. In some preferred embodiments, the marker for positive selection is removed, for example by a site-specific recombinase such as Cre recombinase. Thus, in some embodiments, the non-human animal of the invention is a mouse comprising in its genome at least one allele of a protein C gene, said allele comprising the amino acid sequence of SEQ ID NO:9, and the neomycin resistance gene (Neor, located within SEQ ID NO: 9) has been removed. In embodiments where the marker for positive selection has been removed by a site-specific recombinase (e.g., Cre), the nucleotide sequence of a single site-specific recombination site typically remains in the allele. Preferably, such alleles are substantially as shown in fig. 1D herein.

In preferred embodiments, the only substantial modification to the protein C allele (or protein C gene) in the non-human animal (i.e., a modification compared to the wild-type protein allele in a corresponding non-human animal) is a substitution or substitution. The endogenous nucleotide sequence encoding protein C is modified by a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof. Markers for positive selection and/or the presence of one or more site-specific recombination sites are generally not considered substantial modifications.

Thus, in a preferred embodiment, the human protein C allele according to the invention comprises the sequences upstream (5 '-) and downstream (3' -) of the nucleotide sequence encoding human protein C, or a functional fragment or a functional mutant thereof, of the wild type of the relevant non-human animal. Such sequences may include regulatory sequences, such as promoters and/or enhancers, and the like. Thus, in a preferred embodiment, the expression of the nucleotide sequence encoding human protein C, or a functional fragment or mutant thereof, is under the control of endogenous regulatory sequences of the protein C gene of the relevant non-human animal.

In some alternative embodiments, the human protein C alleles derived from the present invention may additionally comprise one or more regulatory sequences (e.g., promoters and/or enhancers) of the human protein C gene, e.g., in place of one or more regulatory sequences of non-protein C of the non-human animal.

In some embodiments, the genetically modified non-human animal of the invention comprises one or more additional genetic modifications in the genome in addition to the modification with a nucleic acid encoding human protein C or a fragment or mutant thereof. The person skilled in the art is familiar with methods for carrying out such genetic modifications, for example for carrying out gene modifications, gene knockouts.

In some such embodiments, the additional genetic modification is the down-regulation or inactivation (lack or deletion of the animal) of one or more other (non-protein C) genes. For example, the additional genetic modification may be a knock-out of one or more other genes.

In some embodiments, the additional genetic modification may be a gene encoding a coagulation factor, e.g., one or more coagulation factors may be additionally knocked out. In some embodiments, the additional genetic modification may be a gene encoding factor VIII, such as a knock-out of factor VIII. In some embodiments, the additional genetic modification may be a gene encoding factor IX, such as a knock-out of coagulation factor IX. In some embodiments, the additional genetic modification may be a gene encoding factor VIII and factor IX, such as a knock-out of factor VIII and factor IX.

In some embodiments, the non-human animal of the invention is an experimental non-human animal model, such as a mouse model. Such experimental animal models are generally suitable for in vivo studies of human protein C. Such experimental animal models are often particularly suited for testing agents (e.g., candidate therapeutic agents) to identify the potential for using such agents in therapy (e.g., human therapy). In some embodiments, treating (or potentially treating) refers to treating (or potentially treating) a disease or disorder associated with protein C or APC (or protein C or APC pathway), as discussed elsewhere herein.

The invention includes every developmental stage of the non-human animal, such as embryonic, juvenile or adult. The non-human animal in some embodiments is an adult animal. In some embodiments, the mouse is selected for use as a mouse that is at least six weeks old, preferably at least eight weeks old (e.g., 8-10 weeks old).

In addition, the present invention relates to a genetically modified non-human animal (e.g., a mouse) comprising in its genome at least one nucleic acid molecule encoding human protein C or a functional fragment or a functional mutant thereof, which nucleic acid molecule is located in and can be expressed from a protein C gene of the genetically modified non-human animal. Preferably, the at least one nucleic acid molecule is operably linked in the genome of the non-human animal to one or more regulatory elements (e.g. promoter and/or enhancer) of the (endogenous) protein C gene of the non-human animal. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In addition, the invention relates to a non-human animal (e.g., a mouse) having a modified protein C gene site characterized by encoding human protein C (or a functional fragment or functional variant thereof) and not encoding protein C endogenous to the non-human animal. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In addition, the present invention relates to a knock-in non-human animal (e.g., a mouse) whose nucleic acid molecule comprises a nucleotide sequence encoding human protein C or a functional fragment or mutant thereof, which nucleotide sequence encodes human protein C or a functional fragment or mutant thereof knockin one or more copies of the protein C gene in the genome of the non-human animal. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In addition, the present invention relates to a humanized non-human animal (e.g., a mouse) comprising in its genome at least one humanized protein C allele characterized in that the nucleotide sequence encoding endogenous protein C has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In addition, the invention relates to a non-human transgenic animal (e.g., a mouse) in which at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or mutant thereof. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In addition, the present invention relates to genetically modified non-human animals (e.g., mice) whose genome comprises a nucleotide sequence encoding human protein C operably linked to endogenous protein C regulatory sequences (e.g., promoters/enhancers) at the non-human animal's protein C site, or a functional fragment or functional mutant thereof. The embodiments of the other aspects of the invention described apply mutatis mutandis to this aspect of the invention.

In addition, the present invention relates to a non-human animal (e.g., a mouse) carrying (or comprising) a genetically-exchangeable, genetically-modified in nucleotide sequence, the exchange being a replacement (or exchange or substitution) of an endogenous nucleotide sequence encoding a C protein. The genome of the non-human animal consists of a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof. The genetic exchange is established by technical means, according to the discussion elsewhere herein. The embodiments of the other aspects of the invention described herein are applicable to this aspect of the invention mutatis mutandis.

In addition, the present invention relates to a genetically engineered non-human animal (e.g., a mouse) wherein the animal comprises in its genome (e.g., stably integrated into its genome) one or more copies of a nucleotide sequence encoding human protein C or a functional fragment or mutant thereof and is capable of expression. Preferably, such animals do not encode and are unable to express endogenous protein C. Embodiments of other aspects of the invention described herein are applicable to this aspect of the invention by contrast. For example, the nucleotide sequence may be located in a protein C gene of a non-human animal, e.g., replacing a nucleotide sequence encoding endogenous protein C in a non-human animal, as described elsewhere herein.

In addition, the present invention relates to genetically modified non-human animals (e.g., mice) that express or are capable of expressing human protein C or a functional fragment or functional mutant thereof and that do not express or are incapable of expressing endogenous protein C (i.e., do not express or are incapable of expressing non-human animal wild-type or native protein C). The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

Typically, the non-human animals involved in the present invention are fertile and are capable of inheriting the human protein C alleles described in the present invention to their offspring.

Accordingly, the present invention provides a method for the manufacture of a cell or cell line derived from a genetically modified non-human animal of the invention, wherein the cell or cell line comprises in its genome at least one (preferably two) human protein C alleles. The cell may be a somatic cell or a germ cell. In some embodiments, the cell or cell line is a pluripotent stem cell or cell line, such as an embryonic stem cell or cell line derived from a non-human animal or an Induced Pluripotent Stem Cell (iPSC); in some embodiments, the cell or cell line is derived from a cell line of a non-human animal somatic cell. The embodiments of the other aspects of the invention described herein are applicable to this aspect of the invention mutatis mutandis.

In addition, the present invention relates to a tissue or organ derived from a genetically modified non-human animal, wherein said tissue or organ comprises in its cellular genome at least one (preferably two) human protein C alleles. Embodiments of other aspects of the invention are applicable to this aspect of the invention mutatis mutandis.

In addition, the present invention relates to a cell-containing sample derived from a genetically modified non-human animal, wherein said sample comprises at least one (preferably two) human protein C alleles in the genome of its cells. Embodiments of other aspects of the invention are applicable to this aspect of the invention mutatis mutandis.

In addition, the present invention relates to a non-human (e.g., mouse) pluripotent stem cell wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human pluripotent cell has been replaced with a nucleotide sequence encoding human protein C (or a functional fragment or a functional mutant of human protein C). Embodiments of other aspects of the invention are described as being applicable to this aspect of the invention. The non-human pluripotent stem cell may be an embryonic stem cell (ES) or an Induced Pluripotent Stem Cell (iPSC), preferably an embryonic stem cell (ES). The non-human pluripotent stem cell is preferably a mouse pluripotent stem cell, such as a mouse embryonic stem cell (ES) or a mouse Induced Pluripotent Stem Cell (iPSC). Particularly preferred are mouse Embryonic Stem (ES) cells (e.g., C57BL/6ES cells).

In addition, the present invention provides a vector for homologous recombination in a non-human pluripotent stem cell (e.g. an isolated non-human pluripotent stem cell), the vector being capable of replacing (or conferring or mediating replacement of) at least one copy of an endogenous nucleotide sequence encoding protein C or a functional fragment or a functional mutant thereof in the genome of the non-human pluripotent stem cell.

The vectors of the invention may also be used as targeting vectors or gene targeting vectors, recombinant vectors or recombinant targeting vectors. The vector is a nucleic acid molecule, preferably a DNA molecule.

The non-human pluripotent stem cells are preferably derived from a non-human animal species as described elsewhere herein. In some embodiments, the non-human pluripotent stem cells are non-human embryonic stem cells (ES). The non-human pluripotent stem cells are preferably mouse pluripotent stem cells. Particularly preferred are mouse Embryonic Stem (ES) cells. In some embodiments, the mouse Embryonic Stem (ES) cell is a C57BL/6ES cell.

In addition, in some embodiments, the present invention provides a vector comprising in functional combination (i) a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof, preferably a nucleotide sequence encoding human protein C; (ii) at least one marker for positive selection (e.g., an antibiotic resistance gene such as the neomycin resistance gene Neor); (iii)5' -homology arm; (iv)3' -homology arm.

In some embodiments, the marker for positive selection is flanked by site-specific recombination sites (e.g., loxP sites) that are recognized by a recombinase (e.g., Cre recombinase).

In some embodiments, the marker used for positive selection and its flanking specific recombination sites are located in one of the homology arms. In other words, in some embodiments, one homology arm in the vector is interrupted by the marker for positive selection and its flanking specific recombination sites. Thus, in some embodiments, one of the homology arms (e.g., the 3' -homology arm) comprises two different portions or sub-portions, with the marker for positive selection and its flanking specific recombination sites located between the two portions.

In some embodiments, at least one marker for negative selection, e.g., a gene encoding a toxin such as diphtheria toxin a, DTA or a gene encoding thymidine kinase, is additionally present in the vector.

In a preferred embodiment, the vector of the invention comprises, in 5' to 3' order, (i) a 5' -homology arm, (ii) a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant thereof, preferably a nucleotide sequence encoding human protein C; (iii) (iii) a 3 '-homology arm and (iv) a marker for positive selection (which may be flanked by site-specific recombination sites), wherein the marker for positive selection and its flanking site-specific recombination sites are located within the 3' -homology arm. In some such embodiments, a marker for negative selection is additionally present in the vector and is located 5 'corresponding to the 5-homology arm (e.g., at the 5' end of the 5-homology arm).

A homology arm (5 '-or 3' -homology arm) is a portion (or fragment) of DNA whose nucleotide sequence corresponds (or substantially corresponds) to the nucleotide sequence of a genome of interest of a non-human animal. In other words, a homology arm is a polynucleotide whose sequence corresponds (or substantially corresponds) to a nucleotide sequence in the genome of the relevant non-human cell to be targeted. In some embodiments, the homology arm corresponds or substantially corresponds (e.g., has at least 90% or at least 95% or at least 99% identity, preferably 100%) to a portion of a nucleotide sequence in the genome of a corresponding non-human cell.

The homology arms are of sufficient length to confer (or mediate) homologous recombination between the vector and the corresponding nucleotide sequence (or target homologous chromosomal region) in the genome of the non-human animal cell to be targeted. Each homology arm is typically at least 500 base pairs in length (e.g., 1,000 to 10,000 base pairs in length). The length of the 5 '-homology arm and the 3' -homology arm need not be the same.

Upon introduction into a non-human animal cell, the homology arms can undergo homologous recombination with corresponding genomic DNA sequences (targets) in the non-human cell to effect genetic modification of the corresponding (or targeted) chromosomal gene locus. The chromosomal gene locus described in the present invention is a protein C gene, and the genetic modification refers to replacement of an endogenous nucleotide sequence encoding protein C in the genome of a non-human cell with a nucleotide sequence encoding human protein C (or a functional fragment or a functional mutant).

Therefore, the homology arms in the present invention generally consist of nucleotide sequences that can undergo homologous recombination with the corresponding genomic nucleotide sequences of the endogenous protein C gene (or protein C gene site) in the non-human pluripotent stem cell, and allow targeted replacement of the endogenous nucleotide sequences encoding endogenous protein C in the genome of the non-human pluripotent stem cell with the nucleotide sequences encoding human protein C or functional fragments or functional mutants thereof.

The 5 '-homology arm consists of (or consists essentially of) a nucleotide sequence that corresponds or essentially corresponds (e.g., has at least 90% or at least 95% or at least 99% identity, preferably 100%) to the 5' end of a nucleotide sequence that encodes protein C in a non-human animal genome. Thus, typically, upon introduction of the vector into a non-human animal cell, the 5 '-homology arm is capable of (or mediating) homologous recombination with a corresponding (or target) sequence in the non-human animal genome that is located 5' to the nucleotide sequence encoding protein C.

The 3 '-homology arm consists of (or consists essentially of) a nucleotide sequence that corresponds or substantially corresponds (e.g., has at least 90%, or at least 95%, or at least 99% identity, preferably 100%) to the 3' end of an endogenous nucleotide sequence that encodes protein C in the genome of the non-human animal. Thus, typically, upon introduction of the vector into a non-human animal cell, the 3 '-homology arm is capable of (or capable of mediating) homologous recombination with a corresponding (or target) genomic DNA sequence 3' to the nucleotide sequence encoding protein C in the non-human animal genome.

Those skilled in the art are familiar with homology arms, and will be able to readily identify and select suitable homology arms (including vectors of the invention). For example, genomic fragments containing homology arms can be amplified (e.g., using high fidelity Taq DNA polymerase) from commercially available BAC (bacterial artificial chromosome) clones containing genomic sequences required for the relevant non-human animal.

In some embodiments, the 5' -homology arm of the vectors of the invention comprises SEQ ID NO:2, or a sequence substantially homologous thereto (e.g., having at least 90%, alternatively at least 95%, alternatively at least 99% sequence identity to SEQ ID NO: 2) preferably consists of (or consists of): 2, and a 5' homology arm.

In some embodiments, the 3' -homology arm of the vectors of the invention comprises SEQ ID NO:3 and/or the nucleotide sequence of SEQ ID NO:4, or both (e.g., a sequence having at least 90%, 95%, or at least 99% sequence identity and/or substantial homology to SEQ ID NO:3, and/or SEQ ID NO: 4). In some embodiments, the 3' -homology arm of the vectors of the invention comprises SEQ ID NO:3 and the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence identical to SEQ ID NO:3 and/or SEQ ID NO:4 substantially homologous sequence. In some embodiments, it is preferred that the polypeptide comprising SEQ ID NO:3 and SEQ ID NO:4, 3' homology arm of the nucleotide sequence of seq id no.

In some embodiments, the 3' -homology arm comprises SEQ ID NO:3 (or a substantially homologous sequence) and the nucleotide sequence of SEQ ID NO:4 (or a substantially homologous sequence), wherein SEQ ID NO:3 (or a substantially homologous sequence) and the nucleotide sequence of SEQ ID NO:4 (or a substantially homologous sequence) is separated by a nucleotide sequence encoding a positive selection marker (optionally flanked by site-specific recombination sites).

As mentioned above, the vector comprises a nucleotide sequence encoding human protein C or encoding a functional fragment of human protein C or encoding a functional mutant of human protein C. Preferably, the vector comprises a nucleotide sequence encoding human protein C. Amino acid fragments of human protein C are shown herein as SEQ ID NO: 11. thus, preferably, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 11. A preferred nucleotide sequence encoding human protein C (SEQ ID NO: 11) is SEQ ID NO: 8.

as described above, the vector of the present invention may comprise a marker for positive selection. Markers for positive selection are typically nucleotide sequences encoding proteins that confer antibiotic resistance to cells expressing the protein. A positive selection marker enables the selection (or identification) of cells that have been transfected with the vector and are expressing the vector. Markers for positive selection are typically antibiotic resistance genes. The neomycin resistance gene (Neor, Neo cassette) is a preferred positive selection marker, a positive selection marker that is routinely used in the art, is well known, and is well characterized. Expression of the neomycin resistance gene can be selected using the antibiotic G418.

As noted above, in some embodiments of the vectors of the invention, the marker used for positive selection is flanked by generally identical site-specific recombination sites that are recognized by a site-specific recombinase (i.e., there are two generally identical site-specific recombination sites, one located at the 5 'end of the positive selection marker and the other located at the 3' end of the positive selection marker.

Preferably, the site-specific recombination site is a loxP site. The loxP site nucleotide sequence is shown herein as SEQ ID NO: 7. when exposed to an appropriate site-specific recombinase, e.g., Cre recombinase, recognizes the recombination site-specific recombination sites in the presence of LoxP sites and mediates deletion of the nucleotide sequence located between the two site-specific recombination sites, such deleted nucleic acid sequence being a marker for positive selection in the present invention.

In some embodiments, the vector of the invention comprises, in 5' to 3' order, (i) a 5' -homology arm, (ii) a nucleotide sequence encoding human protein C (or encoding a functional fragment of human protein C or encoding a functional mutant of human protein C), preferably a nucleotide sequence encoding human protein C; (iii) a first portion of a 3' homology arm; (iv) a first site-specific recombination site; (v) markers for positive selection; (vi) (vii) a second site-specific recombination site, and (vii) a second portion of the 3' homology arm. Preferably, vectors (i) - (vii) are as described elsewhere herein.

Thus, in a preferred embodiment, the vector of the invention comprises, in order from 5' to 3', (i) a 5' -homology arm comprising the amino acid sequence of SEQ ID NO:2 (or consists thereof); (ii) a nucleotide sequence encoding human protein C; (iii) a first portion of a 3' -homology arm, the first portion comprising SEQ ID NO:3 (or consisting thereof); (iv) a first loxP recombination site comprising SEQ ID NO:7 (or consisting thereof); (v) the positive selection marker is the neomycin resistance gene (Neor); (vi) a second loxP recombination site comprising the sequence of SEQ ID NO:7 (or consisting thereof); (vii) a second portion of the 3' -homology arm, the second portion comprising SEQ ID NO:4 (or consists thereof).

In a preferred embodiment, the vector comprises SEQ ID NO: 9. SEQ ID NO:9 is a nucleotide sequence present in the vector in the examples section herein for transfecting targeted mouse embryonic stem ES cells as described in the present invention, which are then used to make genetically engineered mice according to the present invention. SEQ ID NO:9 comprises a 5' -homology arm, a nucleotide sequence encoding human protein C, a first portion of a 3' -homology arm, a flanking neomycin resistance gene (Neor) flanked by loxP, and a second portion of the 3' -homology arm.

As described above, in some embodiments, a negative selection marker is additionally present in the vector. A negative selection marker is a nucleotide sequence (or gene) that, when expressed in a cell, encodes a protein that causes cell death (or is toxic to the cell). The nucleotide sequence (or gene) encoding the negative selection marker is located outside the homology arm of the vector, i.e., 5 'with respect to the 5' homology arm, or 3 'with respect to the 3' homology arm.

In some embodiments, the negative selection marker is a nucleotide sequence encoding a toxin, such as Diphtheria Toxin A (DTA) or a nucleotide sequence encoding thymidine kinase, preferably a nucleotide sequence encoding DTA. Nucleotide sequences outside the homology arms are usually lost during homologous recombination, but if the vector is randomly integrated (non-homologous recombination) into the genome, the negative selection marker is usually retained and expressed. In this case, unlike the occurrence of the correct homologous recombination event, the negative selection marker is usually transcribed and translated, which is detrimental to the selectivity of clones with non-homologous (random) integration. The negative selection marker DTA is toxic to cells by inhibiting protein synthesis and therefore will usually eliminate cells expressing DTA. Cells expressing the negative selection marker Thymidine Kinase (TK) are sensitive to thymidine analogues. Thus, cells expressing TK can be eliminated (or selectively eliminated) by culture in the presence of thymidine analogs.

In some embodiments, the vectors of the invention comprise, in 5 'to 3' order, (i) a marker for negative selection; (ii) (ii) a 5' -homology arm, (iii) a nucleotide sequence encoding human protein C or encoding a functional fragment of human protein C or encoding a functional mutant of human protein C, preferably a nucleotide sequence encoding human protein C; (iv) a first part of a 3 '-homology arm, (v) a first site-specific recombination site, (vi) a marker for positive selection, (vii) a second site-specific recombination site, and (viii) a second part of a 3' -homology arm. Preferably, elements (i) - (viii) are as described elsewhere herein.

Thus, in a preferred embodiment, the vector of the invention comprises, in order 5 'to 3', (i) a marker for negative selection; (ii) a 5' -homology arm comprising SEQ ID NO:2 (or consists thereof); (iii) a nucleotide sequence encoding human protein C; (iv) a first portion of a 3' -homology arm, the first portion comprising (or consisting of) the amino acid sequence of SEQ ID NO:3, (v) a first loxP recombination site comprising (or consisting of) the nucleotide sequence of SEQ ID NO: 7; (vi) a positive selection marker for the neomycin resistance gene (Neor); (vii) a second loxP recombination site comprising SEQ ID NO:7 (or consisting thereof); and (viii) a second portion of the 3' -homology arm, the second portion comprising SEQ ID NO:4 (or consists thereof).

In one embodiment, the vector comprises SEQ ID NO:9 and has a marker for negative selection located 5' or 3' (preferably located 5 ') thereto.

In addition to the components of the vectors described above that are important for targeting (i.e., the generation of "knock-in" alleles), the vectors typically contain vector backbone nucleotide sequences. Substantial components of the vector can be readily assembled and cloned into the vector backbone using methods conventional in the art (e.g., Green and Sambrook, 2012, Molecular Cloning: A Laboratory Manual, 4th Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) and other Laboratory textbooks. Methods of making vectors for gene targeting are well known in the art, and any suitable method may be used.

In some embodiments, the vector comprises SEQ ID NO:1 or a sequence substantially homologous thereto (or consisting thereof). Substantially homologous sequences are described elsewhere herein. Preferably, if the vector has such a substantially homologous sequence, the nucleotide sequence change is made in the sequence of the targeting vector consisting of SEQ ID NO:9 outside the area defined by the reference numeral. In a preferred embodiment, the vector comprises SEQ ID NO:1 (or consists thereof). The carrier may be circular or linear.

Vectors are typically constructed (or assembled) as circular nucleic acid (DNA) molecules and then linearized prior to use. Thus, in some embodiments, the support is a linearized support. Linearization is generally performed using restriction enzymes that recognize and cleave restriction sites located outside the essential components of the vector (e.g., in the vector backbone). In the 5 'position relative to the 5' homology arm, and in the 3 'position relative to the 3' homology arm. In some embodiments, the vector is linearized using the restriction enzyme NotI. For example, in some embodiments, the vector of the invention is a linearized vector produced by linearizing (e.g., with NotI) a circular vector comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1.

The term "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences, thereby comprising non-naturally occurring monomers or fragment portions thereof. The nucleic acid sequences of the invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. Deoxyribonucleic acid sequence (DNA) sequences are preferred. The sequence may also comprise modified bases. The nucleic acid molecule may be double-stranded or single-stranded, preferably double-stranded. The nucleic acid molecule may be wholly or partially synthetic or recombinant.

The nucleic acid molecules of the invention may be "isolated" or "purified". The term "isolated" or "purified" generally refers to nucleic acids that are substantially free of cellular material, or free of other nucleic acids from which they are derived or from which they are produced.

In another aspect, the invention provides a non-human pluripotent stem cell that has been transfected with a vector of the invention.

In another aspect, the invention provides a method for making (or producing) a non-human pluripotent stem cell in whose genome at least one copy of an endogenous nucleotide sequence encoding protein C is replaced with a nucleotide sequence encoding human protein C or a functional fragment or mutant of human protein C. The embodiments of the other aspects of the invention described herein are applicable to this aspect of the invention mutatis mutandis.

In some embodiments, a method for making (or generating) a non-human pluripotent stem cell comprises the steps of:

(i) transfecting a non-human pluripotent stem cell with the vector of the invention; and

(ii) selecting one or more transfected non-human pluripotent stem cells of (I) to identify in its clonal genome at least one copy of an endogenous nucleotide sequence encoding protein C that has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or mutant of human protein C.

This method of generating non-human pluripotent stem cells is an in vitro method.

Preferably, the non-human pluripotent stem cells are of a non-human animal species as described elsewhere herein. In some embodiments, the non-human pluripotent stem cells are non-human Embryonic Stem (ES) cells. Preferably, the non-human pluripotent stem cells are mouse pluripotent stem cells. Particularly preferably, the non-human pluripotent stem cells are mouse Embryonic Stem (ES) cells. In some embodiments, the mouse Embryonic Stem (ES) cell is a C57BL/6ES cell.

Step (i) of the method involves transfecting a non-human pluripotent stem cell with a vector of the invention. Typically, the vector is linearized (e.g., with NotI) prior to transfection. Transfection may be carried out by any suitable means and the skilled person is familiar with suitable and standard transfection protocols. For example, transfection may be performed by electroporation, lipofection, nuclear transfection, and the like. In some embodiments, electroporation is preferred.

Step (ii) of the method involves selecting one or more transfected non-human pluripotent stem cells of (i) in which at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human pluripotent cells has been replaced with a nucleotide sequence encoding human protein C (or a functional fragment or functional mutant thereof). The selection of step (ii) typically comprises analysis (or screening) for the presence of a desired homologous recombination event (or a desired targeting event) in the transfected non-human pluripotent stem cell, i.e. analysis (directly and/or indirectly) for the presence of a protein C allele in the genome of the non-human pluripotent stem cell that has been correctly targeted by the targeting vector.

Typically, the selection step (ii) comprises selecting one or more transfected non-human pluripotent stem cells based on the expression of the marker for positive selection (the marker for positive selection is typically provided by the targeting vector). Preferably, the marker for positive selection is the neomycin resistance gene (Neor) and the selection agent is G418 (e.g. 200. mu.g/ml). Typically, G418 resistant clones are selected and amplified (e.g. in 96-well plates).

Typically, in addition to selection based on expression of the marker for positive selection, the selection step also includes analysis of the genomic DNA to determine whether the desired homologous recombination event has occurred. Analysis of genomic DNA can be performed using PCR (polymerase chain reaction) based methods and/or by southern blotting and/or DNA sequencing. Preferably, PCR and Southern blot based analyses are performed.

In some embodiments, the PCR-based method comprises performing a PCR reaction in which the template DNA is genomic DNA isolated from the transfected non-human pluripotent stem cell under study (i.e., the potentially targeted non-human pluripotent stem cell), and the design of PCR primers is designed accordingly: if the desired homologous recombination event occurs, a PCR product of the expected size will be produced. An exemplary and preferably PCR-based method is described in the examples section herein (and shown in fig. 4).

In some embodiments, Southern blots are performed to determine (or confirm) whether the desired homologous recombination event (targeting event) has occurred. In this assay, genomic DNA isolated from transfected non-human pluripotent stem cells under study (i.e., potentially targeted non-human pluripotent stem cells) was digested with restriction enzymes (e.g., Bsu36I or EcoNI). In this Southern blot, a probe is used which is capable of hybridizing to a DNA fragment of digested genomic DNA, which fragment is expected to have a certain (predetermined) size if the desired homologous recombination event has occurred. In some embodiments, the probe is capable of hybridizing to a digested genomic DNA fragment comprising (or part of) a positive selection marker (e.g., a probe capable of hybridizing to a neomycin resistance gene or a portion thereof). Exemplary and preferred Southern blotting methods are described in the examples section herein (and depicted in FIG. 7).

In a particularly preferred embodiment, one or more transfected non-human pluripotent stem cells are selected to identify one or more non-human pluripotent cell clones according to step (ii) of the method described above, as described in the examples section herein.

Typically, the non-human pluripotent stem cells produced according to the invention are heterozygous for a human protein C allele according to the invention.

In another aspect, the invention provides a non-human pluripotent stem cell produced by the method of the invention.

In another aspect, the invention also provides the use of a vector of the invention in the production of a non-human pluripotent stem cell wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human pluripotent cell has been replaced by a nucleotide sequence encoding human protein C or a functional fragment or mutant of human protein C. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In another aspect, the invention provides a method of producing (or producing) a genetically modified non-human animal of the invention. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

In some embodiments, the method comprises:

(i) providing a non-human pluripotent stem cell wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human pluripotent stem cell has been replaced with a nucleotide sequence encoding human protein C, or with a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C; and

(ii) generating a genetically modified non-human animal from the non-human pluripotent stem cell.

Methods for producing genetically engineered non-human animals from non-human pluripotent stem cells are well known in the art.

In some embodiments, the non-human pluripotent stem cells of (i) are produced by the method for producing non-human pluripotent stem cells described herein.

In some embodiments, the non-human pluripotent stem cell is a non-human pluripotent stem cell, wherein one copy of an endogenous nucleotide sequence encoding protein C in the genome of the non-human pluripotent cell has been replaced with a nucleotide sequence encoding human, or a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C. Thus, in some embodiments, the non-human pluripotent stem cell is heterozygous for a human protein C allele according to the invention.

Typically and preferably, the step of producing the genetically modified non-human animal of step (ii) comprises

(a) (ii) introducing the one or more non-human pluripotent stem cells of step (i) into pre-implantation embryos of animals of the same species;

(b) transferring the pre-implantation embryo into which one or more of the non-human pluripotent stem cells have been introduced in (a) into a female pseudopregnant non-human animal of the same species; and

(c) identifying a progenitor in the offspring of the female non-human animal of (b); and optionally

(d) Mating the progenitors of (C) and identifying the genomes of their progeny that have had at least one copy of the endogenous nucleotide sequence encoding protein C replaced with a nucleotide sequence encoding human protein C, or a nucleotide sequence encoding a functional fragment of human protein C, or a nucleotide sequence encoding a functional mutant of human protein C.

In some embodiments, in step (a), the one or more non-human pluripotent stem cells and the pre-implantation embryos are each derived from a strain of non-human animals with a different coat color (i.e., the one or more non-human pluripotent stem cells are from a non-human animal with one coat color and the pre-implantation embryos are from a non-human animal of the same species with a different coat color).

In some embodiments, in step (a), the one or more non-human pluripotent stem cells are introduced into the pre-implantation embryo by injection.

In some embodiments, in step (c), the identification of the progenitor animal is achieved by identifying a coat mosaic phenomenon exhibited by the offspring of the female animal. This hair color chimera is a progenitor animal (F-0 generation).

In some embodiments, in step (d), the archery animals are typically mated with non-archery animals of the same species. In some embodiments, in step (d), if the non-human pluripotent stem cell of (a) comprises a marker for positive selection flanked by site-specific recombination sites (e.g., loxP sites), the progenitor animal can be mated with an animal expressing a site-specific recombinase (e.g., Cre recombinase) to remove the marker for positive selection.

Typically, in step (d), the identification of the progeny comprises genotyping the progeny to identify that at least one copy of the endogenous nucleotide sequence encoding protein C in the genome of the progeny has been replaced with a nucleotide sequence encoding human protein C, or a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C. In other words, identifying progeny in step (d) includes identifying those progeny resulting from germline transmission of the non-human pluripotent stem cell genomic DNA of (a) comprising the human protein C allele of the invention.

In some embodiments, the genotype of the progeny is determined by PCR-based methods and/or by DNA sequencing. Suitable methods are known in the art. Preferred PCR-based methods and DNA sequencing methods are described in the examples section herein, which represent preferred embodiments.

In some embodiments, a genetically modified non-human animal produced by the methods of the invention has in its genome one copy of an endogenous nucleotide sequence encoding protein C replaced with a nucleotide sequence encoding human protein C, or a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C. Thus, in some embodiments, the non-human animal is heterozygous for the human protein C allele (i.e., it has one copy of the human protein C allele and one endogenous non-human protein C allele according to the invention). Animals can be identified as such heterozygotes by PCR-based methods, e.g., as described in the examples section herein.

In some embodiments, both copies of an endogenous nucleotide sequence encoding protein C in the genome of the genetically modified non-human animal produced by the methods of the invention are replaced with a nucleotide sequence encoding human protein C, or with a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C. Thus, in some embodiments, the non-human animal is homozygous for the human protein C allele (i.e., it has two copies of the human protein C allele without the endogenous non-human protein C allele according to the invention). Such homozygotes for animals can be identified by PCR-based methods, e.g., as described in the examples section herein.

The preparation of non-human animals that are homozygous for the human C protein allele according to the invention may be carried out by mating and identifying (e.g.by PCR-based genotyping) non-human animals that are heterozygous for the human C protein allele according to the invention, and among the offspring of such mating, those animals that are homozygous for the human C protein allele according to the invention may be identified.

Thus, in some embodiments, the method of producing (or producing) a genetically modified non-human animal of the invention further comprises the steps of: identifying the resulting non-human animals of the invention that are heterozygous for the human C protein allele, mating the heterozygous non-human animals together, and identifying from the mated offspring the offspring that are homozygous for the inventor C protein allele.

Thus, this method results in a genetically modified non-human animal according to the invention, which animal is homozygous for the human protein C allele. Thus, in some embodiments, the present invention provides a method for producing a genetically modified non-human animal, wherein both copies of an endogenous nucleotide sequence encoding protein C in the genome of the non-human animal are replaced by a nucleotide sequence encoding human protein C, or by a nucleotide sequence encoding a functional fragment of human protein C or a functional mutant of human protein C.

In some embodiments, the method of making or obtaining a genetically modified non-human animal of the invention further comprises the additional step or steps of introducing one or more additional genetic modifications into the genome of the non-human animal in addition to replacing the endogenous nucleotide sequence encoding protein C with a nucleotide sequence encoding human protein C or a fragment or mutant thereof.

In some embodiments, the further genetic modification is a modification that results in the down-regulation or inactivation of one or more other non-protein C genes (or causes the animal to be absent or deleted). For example, the further genetic modification may be a knock-out of one or more other genes. In some embodiments, the additional gene is a gene encoding a coagulation factor, e.g., one or more coagulation factors may be additionally knocked out. In some embodiments, the additional gene is a gene encoding factor VIII, e.g., factor VIII can be additionally knocked out. In some embodiments, the additional gene is a gene encoding coagulation factor IX, e.g., a coagulation factor IX can be additionally knocked out. In some embodiments, there may be further genetic modifications in the genes encoding factor VIII and factor IX, e.g., factor VIII and factor IX may be additionally knocked out.

One or more further genetic modifications may be introduced by any suitable means, for example, gene knock-outs. By mating a genetically modified non-human animal according to the invention with an animal of the same species comprising the desired further genetic modification (e.g. having the desired gene knocked out). Thus, in some embodiments, the methods may further comprise mating the genetically modified non-human animal of the invention with an animal of the same species from which factor VIII and/or factor IX has been knocked out. Appropriate mating strategies can be readily devised to generate and select (e.g., by PCR-based genotyping) non-human animals comprising genetic modifications according to the invention (i.e., replacement of at least one endogenous nucleotide sequence encoding protein C with a nucleotide sequence encoding human protein C or a fragment or variant thereof), and additional genetic engineering (e.g., factor VIII and/or factor IX knock-out).

In some embodiments, the non-human animal of the invention further comprises a factor VIII knockout. Mating strategies for generating and selecting (identifying) such non-human animals are described in the examples section herein and represent preferred mating strategies according to the present invention.

Genetically engineered non-human animals, such as mice, comprising one or more additional genes for mating may be produced by any suitable method, for example by preparing non-human embryonic stem cells having the desired genetic modification and making genetically modified non-human animals therefrom. However, in many cases, non-human animals comprising The desired genetic modification (e.g., a factor VIII knockout mouse) are commercially available, for example, from Jackson Laboratory (The US) in The united states.

Particularly preferred methods of making (or generating) the genetically engineered mice of the invention are described in the examples section herein.

In another aspect, the present invention provides genetically modified non-human animals produced by the production methods of the present invention.

In another aspect, the invention provides a method of testing one or more agents (e.g., candidate therapeutic agents or drugs) comprising

(a) Providing a genetically modified non-human animal (e.g., mouse) of the invention; and

(b) administering one or more test agents to the animal.

Generally, such methods identify the potential use of one or more agents in therapy (e.g., to treat a human).

Such test methods may alternatively be considered screening or research methods. Such testing (or screening or research) methods are generally considered preclinical methods. Thus, while these methods aim to determine the potential of an agent in therapy, these test methods are not considered therapeutic methods per se. Thus, in some embodiments, the test method according to the present invention is not a method of treatment.

In a preferred embodiment of the test method of the invention, in the genetically modified non-human animal both copies of the endogenous nucleotide sequence encoding protein C in the genome of the non-human animal have been replaced by a nucleotide sequence encoding human protein C or a functional fragment of human protein C or a functional mutant of human protein C. Thus, in a preferred embodiment, the genetically modified non-human animal of the invention is homozygous for the human protein C allele.

In some embodiments, the test methods according to the present invention further comprise the step of assessing (or determining or evaluating) whether one or more physiological activities or functions, or the extent of change, is present in the animal, and preferably one or more treatment-related changes in physiological activity or function. As discussed elsewhere herein, the relevant physiological activity to be assessed may depend on the particular therapy of interest, i.e., on the particular disease or condition for which a potentially useful therapeutic agent is being tested.

In some embodiments, the alteration of one or more physiological activities (or functions) in the animal is an alteration as compared to a suitable control.

The "control" or "control level" of physiological activity can be the physiological activity in a control animal or a control animal population. One skilled in the art will readily determine the appropriate control for use in the methods of the invention. For example, the control physiological activity (or control physiological activity level) can be a physiological activity of the same species in the transgenic non-human animal of the invention to which the agent (test agent) has not been administered. Other controls can include the physiological activity (or control physiological activity level) of a wild-type (or normal) non-human animal of the same species. The control level can correspond to a level of the same (or equivalent) physiological activity in an appropriate control animal. Alternatively, the control level can correspond to the level of physiological activity discussed in the same individual genetically modified non-human animal measured at an earlier time point (e.g., compared to a "baseline" level for that animal). The control level may also be referred to as a "normal" level or a "reference" level. The control level may be a discrete number or range. Although the control level for comparison may be derived from testing of a suitable control animal or control animal population, as part of the method of the invention, the test method of the invention does not necessarily involve active testing of the control animal, but may involve comparison with a control (or control level) which has previously been determined by the control animal (or control animal population) and is known to the person carrying out the method of the invention.

The alteration may be an increase or a decrease (an increase in physiological activity or a decrease in physiological activity). A change in physiological activity (e.g., as compared to a control) can indicate that the agent is therapeutically or can be useful.

Any measurable (or detectable) change in physiological activity (increase or decrease as the case may be) may indicate that the agent may be therapeutically useful. To indicate that the agent can be used in therapy, the physiological activity is preferably significantly altered compared to a control. More preferably, the level of significant change is statistically significant, preferably with a p-value < 0.05.

In some embodiments, an alteration (optionally an increase or decrease) in the physiological activity (or level of physiological activity) of ≧ 2%,. gtoreq.3%,. gtoreq.5%,. gtoreq.10%,. gtoreq.25%,. gtoreq.50%,. gtoreq.75%,. gtoreq.100%,. gtoreq.200%,. gtoreq.300%,. gtoreq.400%,. gtoreq.500%,. gtoreq.600%,. gtoreq.700%,. gtoreq.800%,. gtoreq.900% or. gtoreq.1,000% compared to the physiological activity (or level of physiological activity) in the appropriate control indicates that the agent may be therapeutically useful.

As described above, the methods of testing agents in the present invention can determine the potential for using such agents in therapy (e.g., human therapy). In a preferred embodiment, the therapy or potential therapy is a therapy or potential therapy for a disease or disorder associated with protein C or APC (or protein C or APC pathway). In some embodiments, the therapy is a treatment for a disease or disorder of an abnormal or deregulated molecular mechanism, molecular pathway or cascade. Wherein protein C or APC (or protein C pathway or APC pathway) is part of (or associated with or involved in regulation of) the molecular mechanism, molecular pathway or cascade. In some embodiments, the therapy is a therapy for a pathophysiological condition involving protein C or APC (or protein C pathway or APC pathway).

In some embodiments, the therapy is the treatment of a bleeding disorder, such as hemophilia (e.g., hemophilia a or hemophilia B) or other disorder characterized by coagulation dysfunction.

In some embodiments, the therapy is a therapy for a disease or condition characterized by inflammation and/or apoptosis (e.g., abnormal or unwanted or excessive inflammation and/or apoptosis), such as sepsis.

In preferred embodiments, the agents to be tested include agents that may alter the level and/or functional activity of human protein C or APC (or protein C or APC pathway). Such agents may include agents from natural sources, such as cell extracts; reagents from synthetic sources, such as compound libraries, or biological libraries (e.g., antibody or peptide libraries).

In a preferred embodiment, the reagents to be tested include reagents that specifically bind or directly bind or interact with human protein C or human APC binding. The agent may include compounds (e.g., small molecule compounds) and antibodies or antigen-binding fragments thereof that bind to or specifically bind to human protein C or human APC.

In a preferred embodiment, the agent to be tested is an antibody or antigen-binding fragment thereof. In a particularly preferred embodiment, the agent to be tested is an antibody that binds (or specifically binds) to human protein C or human APC. Preferably, such antibodies are monoclonal antibodies.

In other embodiments, the agent to be tested is a nucleic acid molecule (e.g., an RNAi, shRNA, or siRNA molecule) that reduces or inhibits translation of human protein C mRNA.

The agents to be tested may include antagonists (inhibitors) and/or agonists (enhancers) of human protein C or human APC (or human protein C or human APC pathway). Whether to screen for an antagonist or possibly an antagonist of human protein C or human APC or to screen for an agent that is an agonist (or possibly an agonist) of human protein C or human APC depends on the particular disease or condition for which a potentially useful therapeutic agent is to be screened, tested or studied or sought.

For example, in embodiments, one method of testing an agent (e.g., a candidate therapeutic or drug) is to determine its potential use in the treatment of hemophilia (e.g., hemophilia a or hemophilia B) or other diseases characterized by impaired clotting function, with the test antagonist (or inhibitor) typically being preferably human protein C or human APC. Such antagonists (or inhibitors) may include antibodies that bind or specifically bind to human protein C or human APC or antigen-binding fragments of such antibodies (antagonist antibodies), or serine protease inhibitors (e.g., small molecule serine protease inhibitors). Preferably an antibody or antigen-binding fragment thereof that binds or specifically binds to human protein C or human APC. Monoclonal antibodies are particularly preferred.

In embodiments, where potential use of a test agent (e.g., a candidate therapeutic or drug) in the treatment of a disease or condition characterized by inflammation and/or apoptosis (e.g., sepsis) is identified, screening for agonists to human protein C or human APC is often preferred.

Typically and preferably, the agent tested is one that is likely to alter the level and/or functional activity of human protein C or human APC (or human protein C or human APC pathway), typically the physiological activity or function assessed is that associated with protein C or APC, or with protein C or APC pathway. Thus, in some embodiments, the physiological activity or function assessed is a physiological activity or function associated with protein C or APC or associated with a protein C or APC pathway. The physiological activity associated with protein C or APC can be any physiological activity of any molecular mechanism or signaling cascade in which protein C or APC is a component and involved. In some embodiments, biomarkers such as the expression (or expression level) of certain genes or proteins can be used as a readout of physiological activity.

Coagulation is the preferred physiological activity. Such physiological activity may be the time required from the start of bleeding to the cessation of bleeding, or the rate of bleeding after the start of bleeding, or the amount of bleeding after the start of bleeding.

Thus, in some embodiments, the physiological activity or function is an activity associated with blood clotting or bleeding. In some embodiments, the physiological activity or function is the time required for blood clotting to occur, e.g., blood clotting time. The time it takes for hemostasis to occur or to stop after bleeding begins, such as injury after bleeding. In some embodiments, e.g., when the transgenic non-human animal is a mouse, the physiological activity or function is the time it takes to stop bleeding or oozing after a tail transection (e.g., a tail distal transection). .

In some embodiments, the time to hemostasis can be determined by administering a test agent to a genetically modified non-human animal described herein, then inducing or inducing bleeding in the animal and measuring the time required for bleeding to hemostasis. If the drug (test drug) has an improvement in hemostasis, for example, a decrease in bleeding cessation time as compared to a control group will generally indicate that the agent can be used to treat hemophilia or other diseases or conditions characterized by clotting dysfunction. The control group may be as discussed elsewhere herein.

In some embodiments, the time required for bleeding can be determined by (i) administering an agent to be tested (e.g., by injection into the orbital vein) to a genetically modified mouse (e.g., an anesthetized mouse) described herein. Optionally, the tail of the mouse is immersed in physiological saline (e.g., at 37 ℃), (ii) the distal tail of the mouse is transected (e.g., at 4 mm) to initiate bleeding (e.g., arterial and venous bleeding), and optionally the tail is immersed in saline (e.g., at 37 ℃), (iii) the bleeding time after tail transection is measured (typically, the time to hemostasis is no less than 1 minute after transection). In some embodiments, the transection of (ii) is performed about 5 minutes after administration of the agent of (i). Particularly preferred methods for determining hemostasis are described in the examples section herein.

In some other embodiments, the hemostasis time can be determined by initiating or inducing bleeding in a genetically modified non-human animal described herein, administering an agent to be tested to the animal, and measuring the time required for bleeding to stop bleeding. If the drug improves hemostasis, for example, a decrease in bleeding cessation time as compared to a control is often indicative of the drug being useful for treating hemophilia or other diseases or conditions characterized by coagulation dysfunction.

In some embodiments, where the physiological activity or function being assessed is an activity associated with blood clotting or bleeding, such as some of the examples described above, the invention can be used to identify potential uses of the agent in treating bleeding disorders, such as hemophilia (e.g., hemophilia a or hemophilia B) or other disorders or conditions characterized by impaired clotting function.

As noted above, if the agent has an improvement in hemostatic effect, for example, a decrease in bleeding cessation time as compared to a control will generally indicate that the agent can be used to treat hemophilia or other diseases or conditions characterized by blood coagulation dysfunction. In some embodiments, a reduction in the time taken for bleeding to stop of ≧ 2%, ≧ 3%, > 5%, > 10%, > 25%, > 50%, > 75%, > 80%, > 90% or 100% as compared to a control indicates that the agent is useful for treating a bleeding disorder such as hemophilia (e.g., hemophilia A or hemophilia B) or other disorder characterized by a coagulation disorder. In some embodiments, a decrease in bleeding cessation time by at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, or at least 1 hour as compared to a control indicates that the medicament can be used to treat bleeding disorders such as hemophilia (e.g., hemophilia a or hemophilia B) or other disorders characterized by impaired clotting function. Suitable controls are discussed elsewhere herein.

In some embodiments, methods are used to test agents to identify potentially useful agents for treating bleeding disorders, such as hemophilia (e.g., hemophilia a or hemophilia B) or other disorders characterized by coagulation dysfunction, wherein the genetically modified non-human animal may lack or lack one coagulation factor, such as coagulation factor VIII or IX. Genetically engineered non-human animals may have one or more genes encoding a coagulation factor knocked out, for example, the genes for coagulation factor VIII and/or coagulation factor IX. Thus, in some embodiments, the genetically modified non-human animals used in such methods may comprise one or more additional genetic modifications that make them deficient or deficient in coagulation factor VIII and/or coagulation factor IX, e.g., they may additionally knock out genes encoding factor VIII and/or encoding factor IX, or may additionally down-regulate or inactivate genes or proteins of factor VIII and/or factor IX. Deficiencies in coagulation factor VIII are characteristic of hemophilia a. Deficiency of coagulation factor IX is a characteristic of hemophilia B.

In some embodiments, the effect of the tested agent on a physiological activity or function (activity associated with coagulation, time taken for bleeding to stop, etc.) can be compared to the effect of a coagulation factor (e.g., coagulation factor VIII or coagulation factor IX) on the same physiological activity. In some embodiments, the coagulation factor (e.g., coagulation factor VIII or coagulation factor IX) may be a human coagulation factor. The coagulation factor may be a recombinant coagulation factor (e.g. a recombinant human coagulation factor) or a coagulation factor purified from plasma (e.g. from human plasma). Thus, in some embodiments, the control physiological activity is a physiological activity in a genetically modified non-human animal according to the invention to which a coagulation factor (e.g., factor VIII or IX) has been administered (positive control).

In some embodiments, when the control physiological activity is that in a genetically modified non-human animal according to the invention to which a coagulation factor (e.g.factor VIII or IX) has been administered, if the tested agent corresponds to ≥ 2%,. gtoreq.3%,. gtoreq.5%,. gtoreq.10%,. gtoreq.25%,. gtoreq.50%,. gtoreq.75%,. gtoreq.80%,. gtoreq.90% or. gtoreq.100% of the activity of the coagulation factor (factor VIII or IX or other procoagulant factor), it may indicate that the medicament is useful for the treatment of a bleeding disorder such as hemophilia (e.g.hemophilia or B hemophilia) or other disorder characterized by impaired coagulation function.

In some embodiments, if the physiological activity (e.g., the time required for hemostasis) of the transgenic non-human animal of the invention to which the test agent has been administered is greater than or equal to 2%, > 3%, > 5%, > 10%, > 25%, > 50%, > 75%, > 80%, > 90% or > 100% of the activity in a control non-human animal of the same species to which the blood coagulation factor has been administered, it may be indicative that the drug is useful for treating a bleeding disorder (e.g., hemophilia A or B)) or other disorder characterized by impaired blood coagulation function.

In some embodiments, if the transgenic non-human animal lacks factor VIII, the physiological activity of the transgenic non-human animal of the invention to which factor VIII has been administered can be used as a control. In some embodiments, if the genetically modified non-human animal lacks coagulation factor IX, the physiological activity of the genetically modified non-human animal of the invention to which coagulation factor IX has been administered can be used as a control.

As noted above, in some embodiments of the methods of testing agents described herein, the objective is to identify agents that may be useful in treating diseases or conditions characterized by inflammation and/or apoptosis, such as sepsis.

In some such embodiments, the physiological activity or function assessed is inflammation or apoptosis. Any suitable means for assessing inflammation or apoptosis may be used, and the skilled person is familiar with suitable methods and assays, for example by challenging the animal with Lipopolysaccharide (LPS) or escherichia coli and assessing the effect of the agent tested thereon. If a reduction in inflammation and/or apoptosis is found in the test drug treated transgenic non-human animal as compared to the control, it is generally indicated that the drug is useful for treating a disease or condition characterized by inflammation and/or apoptosis, such as sepsis.

As mentioned above, the purpose of the test method may be to determine the potential use of a drug in therapy (e.g. human therapy). Treatment includes both treatment and prevention.

In another aspect, the invention provides an agent identified by a test method of the invention. The invention also provides methods of treating a disease or disorder in a subject, preferably a human, such as a human. A disease or condition as defined elsewhere herein, which method comprises administering a therapeutically effective amount of an agent identified by the test method of the invention. The invention also provides an agent identified by a test method of the invention for use in therapy, preferably human therapy, preferably a disease or condition as defined elsewhere herein.

In another aspect, the invention provides the use of a genetically modified non-human animal of the invention in drug screening or drug testing. In another aspect, the invention provides the use of a genetically modified non-human animal of the invention in screening or testing for a candidate therapeutic agent, typically a candidate therapeutic agent that targets or binds human protein C or human APC. Examples of such agents are discussed elsewhere herein.

Such tests are generally considered to be experimental or preclinical. Agents or drugs are often candidate therapeutic agents, but their use in testing is generally not considered therapeutic.

In another aspect, the invention provides the use of a genetically modified non-human animal (e.g., a mouse) as described herein as an experimental animal model. In some embodiments, the experimental model (e.g., a mouse model) is an experimental model of a disease or disorder, preferably a disease or disorder associated with protein C or APC (or a protein C or APC pathway). In some embodiments, the disease or condition is characterized by a disease or condition in which protein C or APC is (or is associated with, or is partially associated with) a molecular mechanism, molecular pathway or cascade that is abnormal or deregulated in the regulation of said molecular mechanism, molecular pathway or cascade. In some embodiments, the disease or condition is a pathophysiological condition involving protein C or APC (or protein C or APC pathway).

In some embodiments, the experimental model is an experimental model (e.g., a mouse model) of a bleeding disorder, such as hemophilia (e.g., hemophilia a or hemophilia B) or other disorder characterized by coagulation dysfunction.

In some embodiments, the experimental model (e.g., a mouse model) is an experimental model of a disease or condition characterized by inflammation and/or apoptosis, such as sepsis.

In some embodiments, the genetically modified non-human animal of the invention is used as an experimental model animal, which may comprise one or more additional genetic modifications, e.g., may comprise a knock-out of one or more additional genes.

In certain embodiments in which the genetically modified non-human animals of the invention are used as experimental models, such as hemophilia or other bleeding diseases or conditions characterized by impaired clotting function, the animals may lack or lack one or more clotting factors. In certain embodiments in which the genetically modified non-human animals of the invention are used as experimental models, such as hemophilia or other bleeding diseases or conditions characterized by impaired clotting function, the animals may lack or lack coagulation factor VIII and/or coagulation factor IX. Thus, in some embodiments in which the genetically modified non-human animal of the invention is used as an experimental model (e.g., hemophilia), the genetically modified non-human animal may comprise one or more additional genetic modifications to make it deficient or deficient in a coagulation factor, e.g., deficient in coagulation factor VIII and/or coagulation factor IX; for example a gene additionally having a gene encoding a knock-out of factor VIII and/or a gene encoding factor IX; for example, to down-regulate or inactivate a gene or protein of factor VIII and/or factor IX.

Where the terms "comprising," "including," "having," or other equivalent terms are used herein, then in some more particular embodiments, these terms include the term "consisting of or" consisting essentially of … … or other equivalent terms.

As used throughout this application, the terms "a" and "an" mean that the components or steps of "at least one," "at least a first," "one or more," or "a plurality" are referred to, unless an upper limit is specifically stated hereinafter.

The invention will now be further described in the following non-limiting embodiments with reference to the accompanying drawings.

FIG. 1 is a wild-type mouse protein C allele (A), targeting vector (B), targeting allele (C) and structural knock-in (KI) allele (Neo)^rAfter deletion) (schematic of D). Elements labeled 1, 2, and 9 are exons. 5' UTR (untranslated)Region) is part of exon 2 of mouse protein C, and the 3' UTR is part of exon 9 of mouse protein C. These UTRs are present in targeting vectors and targeting alleles. Exon 1 contains only the UTR. The human PROC CDS is the coding sequence for human protein C. Neo^r= Neo selection gene cassette. DTA = diphtheria toxin a negative selection marker. Although not depicted in fig. 1A, the wild-type mouse protein C allele also includes

exons

3, 4, 5, 6, 7, and 8 between

exons

2 and 9, as well as intervening introns.

FIG. 2 is an image schematic of a targeting vector.

FIG. 3 shows the digestion of the targeting vector with the indicated restriction enzymes and the separation of the resulting digestion products by gel electrophoresis. The left panel shows the results of digestion with the indicated restriction enzymes (M: marker; 1: ApaL 1; 2: AhdI; 3: FspI/HindIII; 4: NcoI; 5: SacI; 6: NotI). The sizes of the nucleic acid fragments digested with various restriction enzymes are shown in units of kilobase pairs (kb).

Figure 4 is a schematic of a PCR screening strategy for screening for homologous recombination targeting constructs in mouse Embryonic Stem (ES) cells. P1, P2, P3 and P4 are oligonucleotide primers used for PCR screening. The human PROC CDS is the coding sequence for human protein C. Neo^r= Neo selection gene cassette.

FIG. 5 is a gel electrophoresis of the PCR product obtained after 3' arm PCR using primers P1 and P2, as shown in FIG. 4. M: and (4) marking. An expanded view of the marker is shown in the left. WT: the wild type. Various identification numbers (e.g., 2H7, etc.) are the identification numbers of the ES clones being screened.

FIG. 6 is a gel electrophoresis of the PCR product obtained after KI PCR using primers P3 and P4, as shown in FIG. 4. M: and (4) marking. An expanded view of the marker is shown in the left. WT: and (4) a wild type. Various identification numbers (e.g., 2H7, etc.) are the identification numbers of the ES clones being screened.

Figure 7 is a schematic of the Southern blot analysis strategy used to screen for the correct targeting construct in mouse Embryonic Stem (ES) cells. Elements labeled 1, 2, and 9 are exons. The 5'UTR (untranslated region) is part of exon 2 of mouse protein C, while the 3' UTR is mouse protein C exoA part of the display 9. These UTRs are present in targeting vectors and targeted alleles. Exon 1 contains only the UTR. The human PROC CDS is the coding sequence for human protein C. Neo^r= Neo selection gene cassette (also called neomycin resistance gene). EcoNI and Bsu36I are restriction enzymes. Although not depicted in fig. 7A, the wild-type mouse protein C allele also includes

exons

3, 4, 5, 6, 7, and 8 between

exons

2 and 9, as well as intervening introns.

FIG. 8 is the result of Southern blot analysis after digestion of genomic DNA from ES cell clones with the indicated restriction enzymes, electrophoresis of the digestion products and probing with probes hybridized to the Neo selection gene cassette. WT: and (4) a wild type. Various identification numbers (e.g., 2H7, etc.) are the identification numbers of the ES clones being screened.

FIG. 9 is a schematic of the mouse gene identification strategy. F1, F2, F3, F4, R1, R2 and R3 are oligonucleotide primers for PCR-based gene identification screening. Elements labeled 1, 2, and 9 are exons. The 5'UTR (untranslated region) is part of mouse protein C exon 2, while the 3' UTR is part of mouse protein C exon 9. Exon 1 contains only the UTR. The human PROC CDS is the coding sequence for human protein C. Neo^r= Neo selection gene cassette. Although not depicted in fig. 9A, the wild-type mouse protein C allele also includes

exons

3, 4, 5, 6, 7, and 8 between

exons

2 and 9, as well as intervening introns.

FIG. 10 is (A) gel electrophoresis of a PCR product obtained after KI1 PCR using the primers F1 and R1 shown in FIG. 9. (B) Gel electrophoresis of the PCR product obtained after KI2 PCR using primers F2 and R2 as shown in FIG. 9. (C) Gel electrophoresis of PCR products obtained after Neo deletion PCR using primers F3, R3, F4 and R2, as shown in fig. 9. An expanded view of the tag is provided. ESC: embryonic stem cells. WT: and (4) a wild type. MT: mutant alleles (i.e., structural knock-in (KI) alleles after Neo deletion), lanes 1-7 correspond to samples obtained from young animals # 1-7.

FIG. 11 is a gel electrophoresis of the PCR product obtained after PCR using the primers F1 and R3, as shown in FIG. 9. M: and (4) marking. An expanded view of the tag is provided. WT: and (4) a wild type. MT: mutant alleles (i.e., structural knock-in (KI) alleles after Neo deletion).

FIG. 12 is a graph showing bleeding time in a tail-cutting experiment in mice. n = number of mice. WT = wild type. F8-/- (control) F8-/-mice given no octafactors. F8-/- (FVIII) = F8-/-mice given eight factors. Hproc +/+ F8-/- (control) ═ Hproc +/+ F8-/-mice given no octafactor. Hproc +/+ F8-/- (FVIII): hproc +/+ F8-/-mice were given factor eight.

Figure 13 is a schematic of the coagulation cascade. TF = tissue factor. fVII = seven factor. fVIIa = seven factors of activation. fX = ten factor. fXa = ten factors of activation. fVa = five factors of activation. fIX = nine factors. frixa = nine factors activated. fXIa = activated eleven factors. fviia = activated factor eight. Xiii = thirteen factors. Xiiia = activated thirteen factors. APC: activating protein C. PC: protein C. TM: thrombomodulin. (Adapted from Polderdijk and Huntington, 2018,Scientific Reports, 8:8793; adapted under a Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/.）

the embodiment is as follows:

human protein C structural knock-in mouse model

Targeting vector

A targeting vector for knocking-in the human protein C gene into the position of the mouse (C57 BL/6) protein C gene was constructed. The nucleotide sequence of the targeting vector is set forth in SEQ ID NO:1, listed in the table.

The mouse protein C gene (NCBI reference sequence: NM-001042767.3) is located on mouse chromosome 18. 9 exons have been identified, with the ATG of exon 2 being the start codon and the TAG of exon 9 being the stop codon.

The human protein C gene (NCBI reference sequence: NM-000312.3) is located on human chromosome 2. 9 exons have been identified, with the ATG of exon 2 being the start codon and the TAG of exon 9 being the stop codon.

For the knock-in model mice described herein, the region of mouse protein C from the ATG start codon to the TAG stop codon was replaced with the coding sequence of human protein C. Thus, the targeting vector includes the coding sequence of human protein C flanked by homologous, for targeting the homology arms of the mouse protein C gene location to enable targeted replacement of the nucleotide sequence encoding mouse protein C with the human protein C coding sequence.

The targeting vector also includes a Neo selection gene cassette (Neo) located at a 3' position relative to the human protein C coding sequence (human PROC CDS)^r) Flanked by loxP sites. The expression of the Neo selection gene cassette can be selected using the antibiotic G418. The targeting vector also includes a position outside the homology arm of the DTA (diphtheria toxin a) negative selection marker and 5' relative to the 5' end of the 5' homology arm.

Mouse genomic fragments containing Homology Arms (HAs) were amplified from BAC clones using high fidelity Taq DNA polymerase and then assembled into targeting vectors in sequence with site specific (loxP) recombination sites and selectable markers and human protein C coding sequences.

Wild-type mouse protein C alleles, targeting vectors, targeting alleles and structural knock-in (KI) alleles (Neo)^rAfter deletion) is shown in fig. 1.

FIG. 2 provides a further depiction of targeting vectors.

For validation purposes, the targeting vector was digested with restriction enzymes. These results, which confirm restriction enzyme digestion, are shown in FIG. 3. These results demonstrate that the targeting vector is constructed correctly.

The correct construction of the targeting vector was also confirmed by nucleic acid sequencing.

Generation of human protein C structural knock-in mouse Embryonic Stem (ES) cells

The construct targeting human protein C (SEQ ID NO: 1) was linearized by restriction digestion with NotI, followed by phenol/chloroform extraction and ethanol precipitation. The linearized vector was transfected into C57BL/6ES cells according to standard electroporation procedures, and 24 hours after electroporation, the transfected ES cells were subjected to G418 selection (200. mu.g/mL). 564G 418 resistant clones were picked and amplified in 96-well plates. Two copies of the 96-well plate were prepared, one copy was frozen and stored at-80 ℃, and the other copy of the 96-well plate was used for DNA isolation and subsequent PCR screening for homologous recombination. PCR screening identified 16 potential targeted clones, 12 of which were amplified and further characterized by Southern blot analysis. Eight of the twelve amplified clones were confirmed to be correctly targeted. PCR screening and Southern blot analysis are described in more detail below.

The regions shown in FIG. 4 were selected for PCR screening. The PCR screening was as follows:

3’arm PCR

3' arm PCR primers:

Neo-F (P1): 5’-AGGCTGGTAAGGGATATTTGCCTG-3’(SEQ ID NO:13)

3’arm-R (P2): 5’-GAGTGAGCCCAGACCCATAACAAT-3’(SEQ ID NO:14)

the expected PCR product:

wild type: non-targeted: -4.5 kb

Reaction system:

ES cell genomic DNA: 2.0 ul

Forward primer(10 μM): 0.8 ul

Reverse primer(10 μM): 0.8 ul

dNTPs(2.5 mM): 2.4 ul

5X LongAmp Taq Reaction: 4.0 ul

LongAmp Taq DNA Polymerase: 1.2 ul

ddH₂O: 8.8 ul

Total: 20.0 ul

circulation conditions are as follows:

Initial denaturation: 94°C 3min

Denaturation: 94°C 30s

Annealing: 60°C 30s

Extension: 65°C 50s/kb

Additional extension: 65°C 10min

Cycles: 33 x

the results of 3' arm PCR screening are shown in FIG. 5.

Clones that were potentially targeted were further screened by PCR for the presence of knock-in (KI) sites.

Primer:

5’arm-F (P3): 5’-TGGGATTACAAGAAACGCCTCAGAC-3’ (SEQ ID NO: 15)

KI-R (P4): 5’-AGGAGTTGGCACGTTTGCGGAT-3’ (SEQ ID NO:16)

expected PCR products:

wild type: is composed of

Targeting type: 380bp

Reaction system:

ES cell genomic DNA: 1.5 ul

Forward primer(10 μM): 1.0 ul

Reverse primer(10 μM): 1.0 ul

P112 Taq DNA Polymerase: 12.5 ul

ddH₂O: 9.0 ul

Total: 25.0 ul

circulation conditions are as follows:

Initial denaturation: 94°C 3min

Denaturation: 94°C 30s

Annealing: 60°C 30s

Extension: 72°C 30s

Additional extension: 72°C 5min

Cycles: 33 x

storage temperature: 25°C

the results of KI PCR screening are shown in FIG. 6.

Based on PCR screening, samples 2H7, 3B2, 3E2, 3C4, 3D8, 3C11, 4B2, 4a4, 4H3, 4G10, 4a12, 5C5, 5G6, 5G8, 6H3, and 6B5 were shown to be potential targeted ES clones.

Positive clones from the PCR screen (2H 7, 4a12, 4B2, 3C4, 3C11, 5G6, 4a4, 3B2, 6B5, 3D8, 5G8 and 4G 10) were amplified and further characterized by Southern blot analysis. The Southern analysis results are shown in FIG. 7. Genomic DNA was digested with Bsu36I or EcoNI and hybridized using a Neo probe. The Neo probe is expected to detect the following DNA fragments from the target allele in Southern analysis: -10.37 kb (Bsu 36I digest) and-11.39 kb (EcoNI digest).

Expected fragment size of blot: neo probe (containing 5 'arm) -10.37 kb-Bsu36I Neo probe (containing 3' arm) -11.39 kb-EcoNI

Eight out of twelve clones (2H 7, 4B2, 3C4, 3C11, 4a4, 3D8, 5G8, and 4G 10) were confirmed by Southern blot analysis to be correctly targeted. The results of the Southern blot analysis are shown in FIG. 8.

Production of human protein C structural knock-in mice

The targeted ES cell clone 3C4 was injected into C57BL/6 albino embryos, which were then reimplanted into CD-1 pseudopregnant females. Coat color was determined in the initial animals and their germ line transmission was confirmed by breeding with C57BL/6 females and genetic identification of offspring. The Cre mice (i.e., mice expressing Cre recombinase) were mated with F0 (the initial animal) to generate F1 mice in which Neo flanked by loxP sites was deleted^rThe gene cassette is selected. Four male and two female heterozygous target mice were generated from clone 3C 4. More detailed information on the mouse gene identification strategy is as follows:

the regions shown in figure 9 were selected for PCR-based gene identification in mice.

KI1 PCR

KI1 PCR primers:

KI-F (F1): 5’-TGGGATTACAAGAAACGCCTCAGAC-3’ (SEQ ID NO:17)

KI-R (R1): 5’-AGGAGTTGGCACGTTTGCGGAT-3’ (SEQ ID NO:18)

expected PCR products:

wild type: is composed of

Targeted: 380bp

Reaction system:

Mouse genomic DNA: 1.5 ul

Forward primer(10 μM): 1.0 ul

Reverse primer(10 μM): 1.0 ul

Premix Taq Polymerase: 12.5 ul

ddH₂O: 9.0 ul

Total: 25.0 ul

circulation conditions are as follows:

Initial denaturation: 94°C 3min

Denaturation: 94°C 30s

Annealing: 62°C 35s

Extension: 72°C 35s

Additional extension: 72°C 5min

Cycles: 33 x

KI2 PCR

KI2 PCR primers:

KI2-F (F2): 5’-GGCTGTGGGCTCCTTCACAACTAC-3’ (SEQ ID NO:19)

KI2-R (R2): 5’-CAGGTTCTTTTCATAGACTTGGTGTGT-3’ (SEQ ID NO:20)

the expected PCR product:

wild type: is composed of

Targeting type: 324bp

Reaction system:

Mouse genomic DNA: 1.5 ul

Forward primer(10 μM): 1.0 ul

Reverse primer(10 μM): 1.0 ul

Premix Taq Polymerase: 12.5 ul

ddH₂O: 9.0 ul

Total: 25.0 ul

circulation conditions are as follows:

Initial denaturation: 94°C 3min

Denaturation: 94°C 30s

Annealing: 62°C 35s

Extension: 72°C 35s

Additional extension: 72°C 5min

Cycles: 33 x

neo deletion identification PCR

Neo deletion identification PCR primers:

Neo-del-F (F3): 5’-AGGGACCTAATAACTTCGTATAGC-3’ (SEQ ID NO:21)

Neo-del-R (R3): 5’-CCTGTTTGTCCTCCACATTCTACT-3’ (SEQ ID NO:22)

WT-F (F4): 5’-CATCTACACCAAAGTGGGAAGC-3’ (SEQ ID NO:23)

KI2-R (R2): 5’-CAGGTTCTTTTCATAGACTTGGTGTGT-3’ (SEQ ID NO:24)

the expected PCR product:

298 bp of wild type

Target 230bp

Reaction system:

Mouse genomic DNA: 1.5 ul

Forward primer1 (F3) (10 μM): 1.0 ul

Reverse primer1 (R3) (10 μM): 1.0 ul

Forward primer2 (F4) (10 μM): 0.5 ul

Reverse primer2 (R2) (10 μM): 0.5 ul

Premix Taq Polymerase: 12.5 ul

ddH₂O: 8.0 ul

Total: 25.0 ul

circulation conditions are as follows:

Initial denaturation: 94°C 3min

Denaturation: 94°C 30s

Annealing: 62°C 35s

Extension: 72°C 35s

Additional extension: 72°C 5min

Cycles: 33 x

mouse genotyping PCRs results:

seven young mice (1#, 2#, 3#, 4#, 5#, 6# and 7#) derived from clone 3C4 were identified as positive (i.e., human protein C knock-in (KI) allele positive) by identifying KI1, KI2 and Neo deletion PCR as described above, and positive young mice were confirmed again by screening Neo deletion by PCR. The results of Neo deletion PCR confirmed that these young mice were heterozygous for the human protein C knock-in (KI) allele. Neo deletion PCR also confirmed that 3C4 ES cells were heterozygous for the target allele. Note that: one mouse (6 # died. The results of gene identification based on mouse PCR are shown in FIG. 10.

Primers F1 and R3 were also used to perform PCR amplification from mouse DNA and the PCR amplified fragments (including the coding sequence of human protein C (CDS) and mouse UTR sequence) were sequenced and no mutation was found. FIG. 11 shows an image of the electrophoretically amplified PCR fragments on an electrophoretic gel.

Sequencing of the PCR primers:

Seq-F (F1): 5’-TGGGATTACAAGAAACGCCTCAGAC-3’ (SEQ ID NO:25)

Seq-R (R3): 5’-CCTGTTTGTCCTCCACATTCTACT-3’ (SEQ ID NO:26)

the expected PCR product:

wild type N.A.

The size of the product is 2795bp

Reaction system:

DNA: 2.0 ul

Forward primer(10 μM): 0.8 ul

Reverse primer(10 μM): 0.8 ul

dNTPs (2.5 mM): 2.4 ul

5X LongAmp Taq Reaction: 4.0 ul

LongAmp Taq DNA Polymerase: 1.2 ul

ddH₂O: 8.8 ul

Total: 2.0 ul

circulation conditions are as follows:

Initial denaturation: 94°C 3min

Denaturation: 94°C 30s

Annealing: 60°C 3s

Extension: 65°C 50s/kb

Additional extension: 65°C 10min

Cycles: 33 x

production of homozygous human protein C knock-in mice and further production of mice with human protein C knock-in and factor VIII deficiency Symptomatic mice

A. Mated heterozygous human protein C (hproC +/-) mice and 58 offspring mice were genotyped by PCR using the following primers:

hproC 1-F: 5’-TGGGATTACAAGAAACGCCTCAGAC-3’ (SEQ ID NO:27)

hproC 1-R: 5’-AGGAGTTGGCACGTTTGCGGAT-3’ (SEQ ID NO:28)

the expected PCR product: wild type, none, target 380 bp. The hproC primer pair recognizes the human protein C knock-in allele but does not recognize the wild-type (i.e., non-targeted) mouse protein C allele.

mproC -F: 5’-CATCTACACCAAAGTGGGAAGC-3’ (SEQ ID NO:29)

mproC -R: 5’-CAGGTTCTTTTCATAGACTTGGTGTGT-3’ (SEQ ID NO:30)

The expected PCR product: 280bp, the source is wild mouse and human protein C heterozygous mouse. The mproC primer pair can identify mouse protein C alleles (but cannot identify alleles directed against human protein C), so that wild-type mice, heterozygous human protein C knock-in mice, and homozygous human protein C knock-in mice can be identified.

Genotype(s)	Number of mice	Theoretical yield	Actual yield
				hproC+/+	3	25%	5.2%
hproC+/-	39	50%	67.2%
				hproC-/-	16	25%	27.6%

Genotype hproC +/+ indicates that the mouse is homozygous for the human protein C coding sequence (i.e., homozygous for the human protein C "knock-in" allele). Since the targeting vector results in the replacement of the mouse protein C coding sequence by the human protein C coding sequence, it can be concluded that mice of genotype hproC +/+ do not contain the mouse protein C coding sequence. Genotype hproC +/-indicates that the mouse is heterozygous for the human protein C coding sequence (i.e., "knockin" for human protein C). Thus, mice with the hproC +/-genotype contain the coding sequence for human protein C (human protein C "knock-in") and the coding sequence for mouse protein C. Genotype hproC-/-indicates homozygous mouse protein C, not containing the human protein C coding sequence (i.e. not containing the human protein C "knock-in").

B. Male hybrid human protein C (hpROC +/-) mice and 37 offspring mice mated to female factor VIII deficient mice (F8-/-; F8-/-mice completely deficient in factor VIII; F8-/-mice from Jackson laboratories, USA) were genotyped by PCR using the following F8 genotypic identification primers (as well as the mproC and hproC primers described in section A above):

F8-Common：5’-GAG CAA ATT CCT GTA CTG AC-3’ (SEQ ID NO:31)

F8-WT-Forward：5’-TGC AAG GCC TGG GCT TAT TT-3’ (SEQ ID NO:32)

F8-Mut-Forward：5’-TGT GTC CCG CCC CTT CCT TT-3’ (SEQ ID NO:33)

the expected PCR product: WT (+) F8=620bp, Mutant (-) F8=420bp

Genotype(s)	Number of mice	Theoretical yield	Actual yield
				hproC+/-, F8+/-	18	50%	48.6%
hproC-/-，F8+/-	19	50%	51.4%

F8-WT-Forward and F8-Mut-Forward primers were used to distinguish between wild type factor VIII (F8) and mutant factor VIII (F8) alleles.

C.Male hybrid human protein C and hybrid factor VIII (hproC +/-, F8 +/-) mice were genotyped by PCR with female factor VIII deficient mice (F8-/-) and 31 offspring mice. Note that: the F8 (factor VIII) gene is located On the mouse X chromosome. Thus, the male heterozygous F8-deficient mouse does not have the wild type F8 gene, and is mating-wise or the like Identical to homozygous F8-deficient mice.

Genotype of a plant	Number of mice	Theoretical yield	Actual yield
				hproC+/-,F8-/-	17	50%	54.8%
hproC-/-,F8-/-	14	50%	45.2%

D. The 12 offspring mice obtained by mating the heterozygous human protein C mouse (hproC +/-) with the heterozygous human protein C and the heterozygous factor VIII mouse (hproC +/-, F8 +/-), were genotyped by PCR

Genotype(s)	Number of mice	Theoretical yield	Actual yield
				hproC+/+,F8+/-	2	25%	16.7%
hproC+/-,F8+/-; hproC+/-,F8+/+	5	50%	41.7%
				hproC-/-,F8+/-; hproC-/-,F8+/+	5	25%	41.7%

E. The 25 offspring mice obtained were genotyped by PCR using male homozygous human protein C, heterozygous factor VIII mice (hproC +/+, F8 +/-) and female heterozygous human protein C, factor VIII deficient mice (hproC +/-, F8-/-), mated

Genotype of a plant	Number of mice	Theoretical yield	Actual yield
				hproC+/+,F8-/-	12	50%	48%
hproC+/-,F8-/-	13	50%	52%

Hemophilia mouse model

Mice of 8 to 10 weeks of age, including WT mice, mice lacking factor VIII (F8-/-) and double mutant mice lacking factor VIII (hproC +/+, F8-/-) having been anesthetized with 80 mg/kg sodium pentobarbital with the abdomen down and the tail immersed in saline at 37 ℃. The tail of the mouse was excised 4mm (severe injury) and the bleeding was both arterial and venous. The tail of the mouse was excised 4mm (severe injury) and the bleeding was both arterial and venous. Controls without human factor VIII injection were also performed. Tail tip transection and immediate tail immersion in 10 ml saline at 37 ℃ bleeding time was measured. The bleeding time is set at a time of stopping bleeding for at least 1 minute. After 15 minutes, the tail was removed from the saline and the bleeding time measurement was ended.

Results and discussion

Humanized protein C knock-in mice were generated by targeted inactivation of the murine protein C gene with human protein C expression modules as described above. Mice (hproC +/+) are fertile and can be crossed with mouse disease models such as coagulation factor VIII deficiency (F8-/-) or coagulation factor IX deficiency (F9-/-) mice, which can be used for functional studies of human protein C and activated protein C in mice.

A human protein C knock-in and factor VIII deficient (hproC +/+, F8-/-) double mutant mouse was obtained as described above. Normally, these mice appear normal and healthy. If double mutant mice (hproC +/+, F8-/-) were bled by tail-cutting, the bleeding time was prolonged similarly to factor VIII deficient mice (F8-/-), which are characteristic symptoms of hemophilia. Human factor VIII can correct prolonged bleeding in mice lacking factor VIII as well as in doubly mutated mice. The results of the bleeding time study are shown in figure 12.

The bleeding profile observed with double mutant mice (hproC +/+, F8-/-) expressing human protein C (but not mouse protein C) is consistent with factor VIII deficient mice (F8-/-) expressing endogenous mouse protein C (but not including human protein C knockins), which is desirable and advantageous because it indicates that human protein C can functionally complement protein C that has been removed in double mutant mice. These results indicate that mice with human protein C knockins will be a useful model to study hemophilia or other pathophysiological conditions involving protein C pathways. The double mutant model described in this example (hproC +/+, F8-/-) is such a model. These models are invaluable and represent the first mouse model for in vivo testing of therapeutic drug candidates targeting human protein C or APC.

The double mutant mouse model described herein (hproC +/+, F8-/-) can be used as a model to test potential candidate therapeutics targeting human protein C or APC. This double mutant mouse model is particularly useful because factor VIII deficiency is characteristic of hemophilia a (the classic hemophilia), and these mice therefore provide a situational model for hemophilia a subjects. Unlike theory, blood clotting in subjects with a deficiency in factor VIII (e.g., hemophilia a patients) can still occur, albeit at a much slower rate. Although factor VIII is the primary target of APC, in subjects lacking factor VIII, APC can still exert anticoagulant activity through its inhibitory effect on activated factor v (fva). Activated factor V is critical for coagulation and is the primary target of APC. Without wishing to be bound by theory, targeting (inhibiting) human protein C/APC in animals deficient in factor VIII (e.g., hemophilia animals characterized by factor VIII deficiency) may improve coagulation by reducing inhibition of activated factor v (fva). Figure 13 provides a schematic of the coagulation cascade.

Other hproC +/+ knock-in mice of the invention (i.e. in addition to the hproC +/+ F8-/-double mutant described in this example) would also be useful mouse models for in vivo studies of human protein C (or human activated protein C, APC) and for the determination of potential protein C/APC targeting therapeutics. For example, an hproC +/+ knock-in mouse that also lacks factor IX would also be a useful mouse model, as it would represent the case for a hemophilia B model that lacks factor IX.

In the case of factor VIII and/or factor IX deficiency or absence (characterizing certain haemophilia), factor X can still be activated by tissue coagulation factor (TF) and activated factor vii (fviia). In the presence of activated factor x (fxa) and activated factor v (fva), which are essential for coagulation as described above, prothrombin can be activated to thrombin to begin coagulation. Targeting (inhibiting) human protein C/APC, e.g. in animals lacking factor VIII and/or factor IX, may improve coagulation by reducing inhibition of activated factor v (fva).

hproC +/+ knock-in mice that do not contain any further genetic modification (e.g., no knock-out of other genes) would also be useful mouse models for studying human protein C/APC activity in vivo and testing potential candidate therapeutics that target human protein C or APC, not only for the identification of potential hemophilia therapies, but also for the identification of therapeutics useful in other pathophysiological conditions involving the protein C pathway.

Sequence listing

<110> Shanghai Rice blood products GmbH

<120> genetically modified non-human animal and application thereof

<160> 33

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17617

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> nucleotide sequence of SEQ ID NO: 1-targeting vector

<400> 1

cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 60

ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 120

ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata 180

cgactcacta tagggcgaat tggagctcca ccgcccgggc tggttctttc cgcctcagaa 240

gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg 300

ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca 360

atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc 420

acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag 480

cgagctctag gatctgcatt ccaccactgc tcccattcat cagttccata ggttggaatc 540

taaaatacac aaacaattag aatcagtagt ttaacacatt atacacttaa aaattttata 600

tttaccttag agctttaaat ctctgtaggt agtttgtcca attatgtcac accacagaag 660

taaggttcct tcacaaagag atcgcctgac acgatttcct gcacaggctt gagccatata 720

ctcatacatc gcatcttggc cacgttttcc acgggtttca aaattaatct caagttctac 780

gcttaacgct ttcgcctgtt cccagttatt aatatattca acgctagaac tcccctcagc 840

gaagggaagg ctgagcacta cacgcgaagc accatcaccg aaccttttga taaactcttc 900

cgttccgact tgctccatca acggttcagt gagacttaaa cctaactctt tcttaatagt 960

ttcggcatta tccactttta gtgcgagaac cttcgtcagt cctggatacg tcactttgac 1020

cacgcctcca gcttttccag agagcgggtt ttcattatct acagagtatc ccgcagcgtc 1080

gtatttattg tcggtactat aaaacccttt ccaatcatcg tcataatttc cttgtgtacc 1140

agattttggc ttttgtatac ctttttgaat ggaatctaca taaccaggtt tagtcccgtg 1200

gtacgaagaa aagttttcca tcacaaaaga tttagaagaa tcaacaacat catcaggatc 1260

catggcacgc gcttctacaa ggcgctggcc gaagaggtgc gggagtttca cgccaccaag 1320

atctgcggca cgctgttgac gctgttaagc gggtcgctgc agggtcgctc ggtgttcgag 1380

gccacacgcg tcaccttaat atgcgaagtg gacctgggac cgcgccgccc cgactgcatc 1440

tgcgtgttcg aattcgccaa tgacaagacg ctgggcgggg tttgctcgac attgggtgga 1500

aacattccag gcctgggtgg agaggctttt tgcttcctct tgcaaaacca cactgctcga 1560

cattgggtgg aaacattcca ggcctgggtg gagaggcttt ttgcttcctc ttgaaaacca 1620

cactgctcga tttgttagca gcctcgaatc aacccgggcg atcctaggcg atgagatcta 1680

gctgtcgcga agagtggcgc gcctccctgc acagctagtc acaacgaagg aaggcgctta 1740

gggaaccctg gcagcttgca aaacgcaaag ggctacggct gcatcgctct tttccagact 1800

tctcagctgg gagcttctgg cagttttccc gagtcactcc tttctctcac tagctcacaa 1860

agtggccagc tgagtcagaa gcctccttct agtacaggcc tgcctcccac caacgccatc 1920

aatcaggaca agtaaggaag acttctgagt cgcccccccc ccccaccggt caaatagagg 1980

ggacatctta tcactgatgg catcctagat tggtgatata tgtaattatt tttgagtgtg 2040

ctacccacga acaagctata tctgtttatg gttgctgttg ttttggtttt tgttttcttt 2100

taaggttctc atccctcagc cactgcgggc aaaaatgaga ccacatttgc caataagttt 2160

gaacacgctc aaccctctct ttctccctcc ctttctgata gacaattcct tcggtaggca 2220

gaggtgagca atgggcacac ggagccttcc agagctggga tcagaaaacc tcttgtttgt 2280

ttgtctgggg agagggaggt tcggcaccaa gggctaagca aatatttgcg gttatggatt 2340

aacctgactc ccagactgac atggcgctac ctggacgaaa ttgcagtttc tccttggccc 2400

acgcctgtaa gtccccctca ttgcaagact gtgaaggact gtggagggag gggaggggag 2460

gaaagtccag ctgggaggaa ggtgacgttc ttgagctaag gctctccagg cagactgaaa 2520

tgtggggcca aggaaaatga gcgcccaaac tctatctgga ccaaggcgtg ggttccctac 2580

aatccaggtg accatctcga cacatgagat tttgtggatc aagtggacag cagtcaaagg 2640

gttccctatg atcaggaacc atcctcagag caatcttgaa acccagaacc ccactactcc 2700

cctctgccct gttctctaag gtggactcta caattccgga agccagcaaa gccgaagagt 2760

gaggccagga ggggctgtgc agctgggata ggcgggcctg cccacctggt ctgggggaca 2820

ctagaggcct tgtggtttga ctactggttg ggggcagggg ttaaggttcc agagttgggg 2880

ccacataggc ttggccttga agccaaactc tgccctcctc ttaggtgtag gcttgtgaca 2940

agccgcgtat ctcctccaag cctttgggtc ccttcccatg aaatggaggt gagaatattc 3000

atgccttcct cttttaacag tgatcagtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 3060

gtgtgtgtgt gtgtgtgtgt gtggtgtaga gtgtggtttc tggtgttcag ggttgaaccc 3120

agaatcttaa acatgccaag tacgttcttt cctactgaac tgcaaccctc cagtatcctg 3180

tacttgttgt ttgtttgttt gtttgtttgt ttgttcggaa gcacctgtgg tggcacacac 3240

ttacaatcct agtgctagag agcagaaaca agtggatccc ttgggcttgc tggctggcca 3300

gcctacgtga tgagtttcag gcagtgagag accttgtccc aaacaataag gtggaagtaa 3360

gccatgatgg catacccctt tagagctagt actcgagagg cagaagcagg tggagtttaa 3420

gaccagcctg gtctacatag aagttccagg atagccaaaa agtacccaag gccatccaaa 3480

aaaacaaaaa caaaacaaaa cctggaggga aaaaaaaaac cagacaatgc ctggggaagg 3540

atgaaggaca gtcagtcaga ttatccctgg tcaacacgtg tgcacaaatc tgtgcacaca 3600

agaaagagct tcacatgggt tactatttgt tttccaacaa ctcattttta agcccccact 3660

ctctttctct gtttttaaaa aaggtttatt tattttatgt atatgagtac attattgctc 3720

tcttcagaca caccagaaaa ggacataaga ttccattaca gatggttgtg agccaccatg 3780

tggttgctgg aatttgaact caggtcctct ggaagagcag tcggtgctct taaccactga 3840

accatctccc cagcccttcc aacaactctt tatggaagaa acctattcta tccattttat 3900

aaatgacaga actgaggcac ggagcacgta aacatcttgt taaatacctc tctctctctc 3960

tctctctccc cagtaggaaa tggaatttgc cccaggcaat gacttttttt tttttttttt 4020

tgctttcatg tacctagagt aagcccagct ctaaaggcca cgagattgtc tgtctgtgga 4080

ccgtggtgta ccccactccc agacccagct tccacacaga caatgagctc acaaacgtcc 4140

tttactccct tccttccttg cttgctctgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtta 4200

agagatcaac ctcagacatt ttctttagga gctatttacc ttgctttttg agacagggcc 4260

tccggatggc ctggagctag tcacctggag ctggtcaagc aggctagggt ggctggccta 4320

agcaatccac aggatctgct tatctctgcc tccccagtcc tgggattaca agaaacgcct 4380

cagacaccta gatttctgtt ttaattttct atgggttctg gggatctttc ttaagtcttc 4440

aagtatgcac ggaaagggct ttattgacta agatatctcc cctgctcagg aatggccctt 4500

tcattctact ttggagggag ctgggggtgg ggcggggagc agctcagctg tgtgtatcct 4560

tggagcttag aagttctcct cagacaggtg tcagcagctc caggatgtgg cagctcacaa 4620

gcctcctgct gttcgtggcc acctggggaa tttccggcac accagctcct cttgactcag 4680

tgttctccag cagcgagcgt gcccaccagg tgctgcggat ccgcaaacgt gccaactcct 4740

tcctggagga gctccgtcac agcagcctgg agcgggagtg catagaggag atctgtgact 4800

tcgaggaggc caaggaaatt ttccaaaatg tggatgacac actggccttc tggtccaagc 4860

acgtcgacgg tgaccagtgc ttggtcttgc ccttggagca cccgtgcgcc agcctgtgct 4920

gcgggcacgg cacgtgcatc gacggcatcg gcagcttcag ctgcgactgc cgcagcggct 4980

gggagggccg cttctgccag cgcgaggtga gcttcctcaa ttgctcgctg gacaacggcg 5040

gctgcacgca ttactgccta gaggaggtgg gctggcggcg ctgtagctgt gcgcctggct 5100

acaagctggg ggacgacctc ctgcagtgtc accccgcagt gaagttccct tgtgggaggc 5160

cctggaagcg gatggagaag aagcgcagtc acctgaaacg agacacagaa gaccaagaag 5220

accaagtaga tccgcggctc attgatggga agatgaccag gcggggagac agcccctggc 5280

aggtggtcct gctggactca aagaagaagc tggcctgcgg ggcagtgctc atccacccct 5340

cctgggtgct gacagcggcc cactgcatgg atgagtccaa gaagctcctt gtcaggcttg 5400

gagagtatga cctgcggcgc tgggagaagt gggagctgga cctggacatc aaggaggtct 5460

tcgtccaccc caactacagc aagagcacca ccgacaatga catcgcactg ctgcacctgg 5520

cccagcccgc caccctctcg cagaccatag tgcccatctg cctcccggac agcggccttg 5580

cagagcgcga gctcaatcag gccggccagg agaccctcgt gacgggctgg ggctaccaca 5640

gcagccgaga gaaggaggcc aagagaaacc gcaccttcgt cctcaacttc atcaagattc 5700

ccgtggtccc gcacaatgag tgcagcgagg tcatgagcaa catggtgtct gagaacatgc 5760

tgtgtgcggg catcctcggg gaccggcagg atgcctgcga gggcgacagt ggggggccca 5820

tggtcgcctc cttccacggc acctggttcc tggtgggcct ggtgagctgg ggtgagggct 5880

gtgggctcct tcacaactac ggcgtttaca ccaaagtcag ccgctacctc gactggatcc 5940

atgggcacat cagagacaag gaagcccccc agaagagctg ggcaccttag cacccctccc 6000

tgctcacctc tggaccctag aagtcactct tggagtaagg ctgggctagt gagtaccaag 6060

acagaggaca ttaaaggagc atgcaacaaa catacctccc cgagtacctg tctgtctttt 6120

catccttttt atgggctatt ctgggggaaa gtaacattaa ttgagcatgc actacacacc 6180

aagtctatga aaagaacctg cttaactccc aaagcagttg tgtagaagat ctagtgggat 6240

ctgagctgat atcacttctg ggggtgagtg gaggagattg atttagagaa aggaattttt 6300

ttagaagtta ctgtaagaga ctaatagagc ctttctcagg gccttggaaa gagcccgtgc 6360

tagttacatc agaaaagctt gccagtgacc agtggccagt gagactcaga atggccatgt 6420

ggtggagcca ggattcaaac caaggtcaca ctcccaaact cagctgcttc tcttctttat 6480

tatccctggg tgtgtgctgg tgtgtgtgtg cgcgcgtggg tgtgtgggtg gatacatgca 6540

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgttatatgt ttggagacca 6600

gaggacaact tcgtttctca acaccatcca cttgttttgt tttgtgtttt gttttgtttg 6660

ttgacacagg gtctctcact gtcctgaaat ctacccagta ggctaggctg gctggctacc 6720

aaaccccacc ccaccctggc tttgacaagt ggagacagaa gaccagtagt ccactggaga 6780

tgtgaccaga tgcccagaag gtgctcctca tggtgcccta cagttttgtt gaggagtctg 6840

tttaataatg cagctgggtg cagtggcagc acctgtagcc cccaatactg aggcagcatt 6900

gctgcagtct gagaggtggg gctcgaggga cctaataact tcgtatagca tacattatac 6960

gaagttatat taagggttcc gcaagctcta gtcgagcccc agctggttct ttccgcctca 7020

gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc 7080

ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag acaatgcgat 7140

gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa 7200

ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat 7260

cagcgagctc tagagaattg atcccctcag aagaactcgt caagaaggcg atagaaggcg 7320

atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg 7380

ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc 7440

acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc 7500

ggcaagcagg catcgccatg ggtcacgacg agatcatcgc cgtcgggcat gcgcgccttg 7560

agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga 7620

tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg 7680

tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg 7740

gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc 7800

aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg 7860

cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg 7920

gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg 7980

gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa 8040

gcggccggag aacctgcgtg caatccatct tgttcaatgg ccgatcccat ggtttagttc 8100

ctcaccttgt cgtattatac tatgccgata tactatgccg atgattaatt gtcaacaggc 8160

tgcaggtcga aaggcccgga gatgaggaag aggagaacag cgcggcagac gtgcgctttt 8220

gaagcgtgca gaatgccggg cctccggagg accttcgggc gcccgccccg cccctgagcc 8280

cgcccctgag cccgcccccg gacccacccc ttcccagcct ctgagcccag aaagcgaagg 8340

agcaaagctg ctattggccg ctgccccaaa ggcctacccg cttccattgc tcagcggtgc 8400

tgtccatctg cacgagacta gtgagacgtg ctacttccat ttgtcacgtc ctgcacgacg 8460

cgagctgcgg ggcggggggg aacttcctga ctaggggagg agtagaaggt ggcgcgaagg 8520

ggccaccaaa gaacggagcc ggttggcgcc taccggtgga tgtggaatgt gtgcgaggcc 8580

agaggccact tgtgtagcgc caagtgccca gcggggctgc taaagcgcat gctccagact 8640

gccttgggaa aagcgcctcc cctacccggt agaatttcga cgacctgcag ccaagctagc 8700

ttcgcgagct cgaccgaaca aacgacccaa cacccgtgcg ttttattctg tctttttatt 8760

gccgctcagc tttacagtga caatgacggc tggcgactga atattagtgc ttacagacag 8820

cactacatat tttccgtcga tgttgaaatc ctttctcata tgtcaccata aatatcaaat 8880

aattatagca atcatttacg cgttaatggc taatcgccat cttccagcag gcgcaccatt 8940

gcccctgttt cactatccag gttacggata tagttcatga caatatttac attggtccag 9000

ccaccagctt gcatgatctc cggtattgaa actccagcgc gggccatatc tcgcgcggct 9060

ccgacacggg cactgtgtcc agaccaggcc aggtatctct gaccagagtc atccttagcg 9120

ccgtaaatca atcgatgagt tgcttcaaaa atcccttcca gggcgcgagt tgatagctgg 9180

ctggtggcag atggcgcggc aacaccattt tttctgaccc ggcaaaacag gtagttattc 9240

ggatcatcag ctacaccaga gacggaaatc catcgctcga ccagtttagt tacccccagg 9300

ctaagtgcct tctctacacc tgcggtgcta accagcgttt tcgttctgcc aatatggatt 9360

aacattctcc caccgtcagt acgtgagata tctttaaccc tgatcctggc aatttcggct 9420

atacgtaaca gggtgttata agcaatcccc agaaatgcca gattacgtat atcctggcag 9480

cgatcgctat tttccatgag tgaacgaacc tggtcgaaat cagtgcgttc gaacgctaga 9540

gcctgttttg cacgttcacc ggcatcaacg ttttcttttc ggatccgccg cataaccagt 9600

gaaacagcat tgctgtcact tggtcgtggc agcccggacc gacgatgaag catgtttagc 9660

tggcccaaat gttgctggat agtttttact gccagaccgc gcgcctgaag atatagaaga 9720

taatcgcgaa catcttcagg ttctgcggga aaccatttcc ggttattcaa cttgcaccat 9780

gccgcccacg accggcaaac ggacagaagc attttccagg tatgctcaga aaacgcctgg 9840

cgatccctga acatgtccat caggttcttg cgaacctcat cactcgttgc atcgaccggt 9900

aatgcaggca aattttggtg tacggtcagt aaattggaca ccttcctctt cttcttgggc 9960

atggccgcag gaaagcagag ccctgaagct cccatcaccg gccaataaga gccaagcctg 10020

cagtgtgacc tcatagagca atgtgccagc cagcctgacc ccaagggccc tcaggcttgg 10080

gcacactgtc tctaggaccc tgagagaaag acatacccat ttctgcttag ggccctgagg 10140

atgagcccag gggtggcttg gcactgaagc aaaggacact ggggctcagc tggcagcaaa 10200

gtgaccagga tgctgaggct ttgacccaga agccagaggc cagaggccag gacttctctt 10260

ggtcccagtc caccctcact cagagcttta ccaatgccct ctggatagtt gtcgggtaac 10320

ggtggacgcc actgattctc tggccagcct aggacttcgc cattccgctg attctgctct 10380

tccagccact ggctgaccgg ttggaagtac tccagcagtg ccttggcatc cagggcatct 10440

gagcctacca ggtccttcag tacctcctgc cagggcctgg agcagccagc ctgcaacacc 10500

tgcctgccaa gcagagtgac cactgtgggc acaggggaca cagggtgggg cccacaacag 10560

caccattgtc cacttgtccc tcactagtaa aagaactcta gggttgcggg gggtggggga 10620

ggtctctgtg aggctggtaa gggatatttg cctggcccat ggagctagct tggctggacg 10680

taaactcctc ttcagaccta ataacttcgt atagcataca ttatacgaag ttatattaag 10740

ggttattgaa tatgatcgga attgggctgc aggaattcga tagcttggct gcaggtcgac 10800

gtacgtagca agcttgatgg gccctggtac cacagcctgg gcaacacagc aaaaatccct 10860

tcccttaaaa aaaacaaaag agaaggaaga aggacgaagt agaatgtgga ggacaaacag 10920

gggagagagg gggaaagaaa gggagggaat tgtcttagag ttttacggct gtgcacagac 10980

accatgatca aggtaactct tgtaaggata acatttagtt ggggctggct tacaggttca 11040

gaagttcagt ccattatcat caaggcagga acatggcagc attcaaggca gacatggtgc 11100

aggaggagct gagagttcta catcttcatc tgaagatttc tagtagaata ctggcttcca 11160

ggcagctagg atgagggtct taaagcccac acccagtgac acacctactc caacagggcc 11220

acacctccta atcatgccac tccctgggct gagcatatag aaaccatcac agagtctaac 11280

tagtgtggcc catcctgcac ccatggaaga ccatcactgg ggcatagaca acctccagag 11340

cccaccctga cagttcctgt ctctgccttc tccagcagtc accagtttca aatagctcct 11400

caaggacaga tggggccttg tgagcttcac cccgctgcag gctggaatgc gccaccttta 11460

atcccagcac ttggaaggca gaggcaggca gatttctgag ttcgaggcca gcctagtcta 11520

cagagtgagt tccaggacag ccagggcgat acagagaaac cctgtctcaa aaaacaaaac 11580

aaaacaaaac gatagaaaag agcaaagtga ccttgggcta tggatgggat ggaccatcgg 11640

gcactgggtt gggaagctga actggtccag atgcccagag cccagagctc tctcctcagc 11700

agttcataac ctggggtgtt gccacagcac acacagcaag gttagttctg ctggttgtcg 11760

ggacttaggg taggaggagt agaagcctgc tactgattct gtctctctct gtttctctct 11820

cctccctccc tccctccttc cctccctccc tccctcttct tcttcttctc atcctcctcc 11880

ctcttcctct tcttcctcca cctccccacc ccttattttg atacagggtt tctctgtgta 11940

tccctggctg tcctggaact cactctgtag cccaggtggg cctcgaactc tcagcctcag 12000

tccccagaat gctgagaaca caggtctgag tgatcactga tggctaaaag ttgggattac 12060

attgttgttg cttgtttgtt tattcttttg tacatgggac ccaaatacaa atagtagcct 12120

caacaataaa cacgggataa gttgctgctc tgctttaggg tctccctgac ctctgttttt 12180

ttgttttttg tttttggtat gttttgtttt ctgttgttgt tgttatcatg tctataaatc 12240

tatcttcctt cctccctcct tccttccctt cctacttccc tctctttctt catccctccc 12300

ttccccctac tctctttcac ccccagatag gaagcaagca tgataaaaac gtgtggtgtt 12360

ttccttttta tgtagagagt actgtgtagt gagtgttatc ctatgggtgc tgccattctg 12420

ctgtatgtta cctgctgtat gttataccaa cctagatggt ggtgaactca catggttgct 12480

tccatcttgg tgaggttacc agccaagatg tctgccctcc acatgattgc ctccatcttg 12540

gtgaggtaaa tgtttaataa agtaacaaaa caagacatta aaaacaaaca ttccagcaca 12600

aaatcttctt gagattagag acataatagg aagtcaggtg aggtcataca ggccattaat 12660

cccatcactt ggggaacaga ggcaggtaga actttgaatt gaaggcaggt atatttagtg 12720

atttccaggg ctatgtagag aggcccctga cccaactaat aagtaaacag gaagataaat 12780

aaatataatg aacttagaat aaaacaaaga aaggaagaaa caagggaagg cagtgctggg 12840

ggcctggctt atggtggatg ggggaattct gtgctagggt gcctgaaact ctgggctcca 12900

tcctctgtag tgcataaact ctttggtacg ttagcccctg tctgtaacaa ggagctgtcc 12960

acggttgcag tactgccttt cccatctcag ctgcccctca ggagctgtcc acagtggcga 13020

cactttttca tcctcagcct acagctttag ggaaacacca ctgcaggggc tgtcccaaag 13080

gtgctgtcca cagaggcagc accttctctg tggtctcacc cctccagaca ccccccagca 13140

gccccacagg gatggcacct cagtaaaagc caactgtggc cagagaagtc ttcctaccct 13200

aactcataga ctcgatgcag ggaaaacagg gtgaaaaaaa gccaccaagc cctgagctcc 13260

ccccagctca ggacttaaaa tctcatcaat cctcactatg gaaatctctg ccttgagaag 13320

ctctgccccc tcataaatcc tatataagaa ctgtcccttt gtccagttcc ctgccatccg 13380

ctcccaggag cagagggcag ttatccctgg attcatccct ccacaccctg gacctgccaa 13440

taaacctttc ttgagatttc atgcttcctg tgattctcag tggaagaagc caagaagaaa 13500

agaaccaaag agagggagcc aggctgaggc tcctgagttc ttcagctcag ctgtggatac 13560

ctgtgatggt ttgtatatgc tctgtccagg gaatggcatg attagaaggt gtggccctgt 13620

tgaagtaggt gtgtcactgt gggtgtgggc tataagaccc tcatcttaac tgcatggaag 13680

tcattcttcc actggcagcc ttcagatgaa aatgtagaac tctcagctcc tcctgcacca 13740

tgcctgccta gatgctgccc tgctcccacc ttgatgataa tggactgaac ctctgaacct 13800

gtaagccagc cccaattaaa tgttgttctt tataagtctt gccttggttg tggtgtctgt 13860

tcacagcagt aaaaccatga ctaagacaat atcttctact tggagctgca acaactctgc 13920

tgaggaggct tcctctcaga gctatatggt tcctggtatc tgtaaaattc ccttctattg 13980

ggacactttc caacctcaga tctgtgtggt tcctggcccc tgtgtctcgg gatgcccttc 14040

cattagaaca gcttctcccc tcagagctgc atggttcctg gacttctgag tcccaggaga 14100

cccttccatc tgagcagaaa cacttacagg agcagagtcc tccaacacca cggttttgtt 14160

ttgaaagacc aagaccaacc ctcaggaggt ttctggcaaa ggcagattct aggtgcacct 14220

ggaggagacc tatagtgcag gaccatccgt cgtaggttgc taggcaccaa tgggcaaagg 14280

tagggaagaa atcttaccag aagattctat tccattccat tctcctcaca atgtaagagc 14340

caaagttaac ctctaaggcc caagaacaag gtaactctcc agaatgctgg gagatgtagt 14400

tcttgggtaa caacaagcca tgttctcgcc ctaaacaagt ttgtttgaat caactacact 14460

gaatgtactt gatcatatgt aggagagaga agattgattc tagttcaggg tttcaggcta 14520

tttcagtcca ccatgatggg aaaggcatga cattgtttat gacagtaaga gcatgtagca 14580

gaggatcctc acatcacaac aggccgaaat gcagaggaca gtgcaaccag aggacagtct 14640

gtaactttcc aagtccctct tctagtgggt tgcttccacc acctctcagg tggtgctaca 14700

gctcaggaac aattgagatg tgtgatgaag ggcaggtact caactgtggc tgtattgtat 14760

ccttttatag ttgtcctctg tgtgttgagc tatgtgcgag attctcaggt catcggagta 14820

cctgttttac tttggcaggc ataggagact cctgagaact ctgcctgaca tccttgccag 14880

cccaagcttt ggtttagtgt gtgcagtatc actcttgggt cttatctgca tatccctgat 14940

ggcccatcaa gatgtgtgcg gccgcgtacc agcttttgtt ccctttagtg agggttaatt 15000

tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 15060

attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 15120

agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 15180

tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 15240

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 15300

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 15360

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 15420

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 15480

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 15540

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 15600

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 15660

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 15720

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 15780

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 15840

cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 15900

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 15960

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 16020

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 16080

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 16140

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 16200

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 16260

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 16320

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 16380

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 16440

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 16500

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 16560

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 16620

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 16680

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 16740

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 16800

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 16860

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 16920

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 16980

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 17040

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 17100

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 17160

aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 17220

cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 17280

tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 17340

gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 17400

tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 17460

ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 17520

tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 17580

taacaaaaat ttaacgcgaa ttttaacaaa atattaa 17617

<210> 2

<211> 2901

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> 5' homology arm of SEQ ID NO 2-targeting vector

<400> 2

tccctgcaca gctagtcaca acgaaggaag gcgcttaggg aaccctggca gcttgcaaaa 60

cgcaaagggc tacggctgca tcgctctttt ccagacttct cagctgggag cttctggcag 120

ttttcccgag tcactccttt ctctcactag ctcacaaagt ggccagctga gtcagaagcc 180

tccttctagt acaggcctgc ctcccaccaa cgccatcaat caggacaagt aaggaagact 240

tctgagtcgc cccccccccc caccggtcaa atagagggga catcttatca ctgatggcat 300

cctagattgg tgatatatgt aattattttt gagtgtgcta cccacgaaca agctatatct 360

gtttatggtt gctgttgttt tggtttttgt tttcttttaa ggttctcatc cctcagccac 420

tgcgggcaaa aatgagacca catttgccaa taagtttgaa cacgctcaac cctctctttc 480

tccctccctt tctgatagac aattccttcg gtaggcagag gtgagcaatg ggcacacgga 540

gccttccaga gctgggatca gaaaacctct tgtttgtttg tctggggaga gggaggttcg 600

gcaccaaggg ctaagcaaat atttgcggtt atggattaac ctgactccca gactgacatg 660

gcgctacctg gacgaaattg cagtttctcc ttggcccacg cctgtaagtc cccctcattg 720

caagactgtg aaggactgtg gagggagggg aggggaggaa agtccagctg ggaggaaggt 780

gacgttcttg agctaaggct ctccaggcag actgaaatgt ggggccaagg aaaatgagcg 840

cccaaactct atctggacca aggcgtgggt tccctacaat ccaggtgacc atctcgacac 900

atgagatttt gtggatcaag tggacagcag tcaaagggtt ccctatgatc aggaaccatc 960

ctcagagcaa tcttgaaacc cagaacccca ctactcccct ctgccctgtt ctctaaggtg 1020

gactctacaa ttccggaagc cagcaaagcc gaagagtgag gccaggaggg gctgtgcagc 1080

tgggataggc gggcctgccc acctggtctg ggggacacta gaggccttgt ggtttgacta 1140

ctggttgggg gcaggggtta aggttccaga gttggggcca cataggcttg gccttgaagc 1200

caaactctgc cctcctctta ggtgtaggct tgtgacaagc cgcgtatctc ctccaagcct 1260

ttgggtccct tcccatgaaa tggaggtgag aatattcatg ccttcctctt ttaacagtga 1320

tcagtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1380

gtgtagagtg tggtttctgg tgttcagggt tgaacccaga atcttaaaca tgccaagtac 1440

gttctttcct actgaactgc aaccctccag tatcctgtac ttgttgtttg tttgtttgtt 1500

tgtttgtttg ttcggaagca cctgtggtgg cacacactta caatcctagt gctagagagc 1560

agaaacaagt ggatcccttg ggcttgctgg ctggccagcc tacgtgatga gtttcaggca 1620

gtgagagacc ttgtcccaaa caataaggtg gaagtaagcc atgatggcat acccctttag 1680

agctagtact cgagaggcag aagcaggtgg agtttaagac cagcctggtc tacatagaag 1740

ttccaggata gccaaaaagt acccaaggcc atccaaaaaa acaaaaacaa aacaaaacct 1800

ggagggaaaa aaaaaaccag acaatgcctg gggaaggatg aaggacagtc agtcagatta 1860

tccctggtca acacgtgtgc acaaatctgt gcacacaaga aagagcttca catgggttac 1920

tatttgtttt ccaacaactc atttttaagc ccccactctc tttctctgtt tttaaaaaag 1980

gtttatttat tttatgtata tgagtacatt attgctctct tcagacacac cagaaaagga 2040

cataagattc cattacagat ggttgtgagc caccatgtgg ttgctggaat ttgaactcag 2100

gtcctctgga agagcagtcg gtgctcttaa ccactgaacc atctccccag cccttccaac 2160

aactctttat ggaagaaacc tattctatcc attttataaa tgacagaact gaggcacgga 2220

gcacgtaaac atcttgttaa atacctctct ctctctctct ctctccccag taggaaatgg 2280

aatttgcccc aggcaatgac tttttttttt ttttttttgc tttcatgtac ctagagtaag 2340

cccagctcta aaggccacga gattgtctgt ctgtggaccg tggtgtaccc cactcccaga 2400

cccagcttcc acacagacaa tgagctcaca aacgtccttt actcccttcc ttccttgctt 2460

gctctgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttaaga gatcaacctc agacattttc 2520

tttaggagct atttaccttg ctttttgaga cagggcctcc ggatggcctg gagctagtca 2580

cctggagctg gtcaagcagg ctagggtggc tggcctaagc aatccacagg atctgcttat 2640

ctctgcctcc ccagtcctgg gattacaaga aacgcctcag acacctagat ttctgtttta 2700

attttctatg ggttctgggg atctttctta agtcttcaag tatgcacgga aagggcttta 2760

ttgactaaga tatctcccct gctcaggaat ggccctttca ttctactttg gagggagctg 2820

ggggtggggc ggggagcagc tcagctgtgt gtatccttgg agcttagaag ttctcctcag 2880

acaggtgtca gcagctccag g 2901

<210> 3

<211> 931

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO: 3-first part of 3 'homology arm of targeting vector (i.e., 5' part)

<400> 3

cacccctccc tgctcacctc tggaccctag aagtcactct tggagtaagg ctgggctagt 60

gagtaccaag acagaggaca ttaaaggagc atgcaacaaa catacctccc cgagtacctg 120

tctgtctttt catccttttt atgggctatt ctgggggaaa gtaacattaa ttgagcatgc 180

actacacacc aagtctatga aaagaacctg cttaactccc aaagcagttg tgtagaagat 240

ctagtgggat ctgagctgat atcacttctg ggggtgagtg gaggagattg atttagagaa 300

aggaattttt ttagaagtta ctgtaagaga ctaatagagc ctttctcagg gccttggaaa 360

gagcccgtgc tagttacatc agaaaagctt gccagtgacc agtggccagt gagactcaga 420

atggccatgt ggtggagcca ggattcaaac caaggtcaca ctcccaaact cagctgcttc 480

tcttctttat tatccctggg tgtgtgctgg tgtgtgtgtg cgcgcgtggg tgtgtgggtg 540

gatacatgca tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgttatatgt 600

ttggagacca gaggacaact tcgtttctca acaccatcca cttgttttgt tttgtgtttt 660

gttttgtttg ttgacacagg gtctctcact gtcctgaaat ctacccagta ggctaggctg 720

gctggctacc aaaccccacc ccaccctggc tttgacaagt ggagacagaa gaccagtagt 780

ccactggaga tgtgaccaga tgcccagaag gtgctcctca tggtgcccta cagttttgtt 840

gaggagtctg tttaataatg cagctgggtg cagtggcagc acctgtagcc cccaatactg 900

aggcagcatt gctgcagtct gagaggtggg g 931

<210> 4

<211> 4126

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO: 4-second part of 3 'homology arm of targeting vector (i.e., 3' part)

<400> 4

acagcctggg caacacagca aaaatccctt cccttaaaaa aaacaaaaga gaaggaagaa 60

ggacgaagta gaatgtggag gacaaacagg ggagagaggg ggaaagaaag ggagggaatt 120

gtcttagagt tttacggctg tgcacagaca ccatgatcaa ggtaactctt gtaaggataa 180

catttagttg gggctggctt acaggttcag aagttcagtc cattatcatc aaggcaggaa 240

catggcagca ttcaaggcag acatggtgca ggaggagctg agagttctac atcttcatct 300

gaagatttct agtagaatac tggcttccag gcagctagga tgagggtctt aaagcccaca 360

cccagtgaca cacctactcc aacagggcca cacctcctaa tcatgccact ccctgggctg 420

agcatataga aaccatcaca gagtctaact agtgtggccc atcctgcacc catggaagac 480

catcactggg gcatagacaa cctccagagc ccaccctgac agttcctgtc tctgccttct 540

ccagcagtca ccagtttcaa atagctcctc aaggacagat ggggccttgt gagcttcacc 600

ccgctgcagg ctggaatgcg ccacctttaa tcccagcact tggaaggcag aggcaggcag 660

atttctgagt tcgaggccag cctagtctac agagtgagtt ccaggacagc cagggcgata 720

cagagaaacc ctgtctcaaa aaacaaaaca aaacaaaacg atagaaaaga gcaaagtgac 780

cttgggctat ggatgggatg gaccatcggg cactgggttg ggaagctgaa ctggtccaga 840

tgcccagagc ccagagctct ctcctcagca gttcataacc tggggtgttg ccacagcaca 900

cacagcaagg ttagttctgc tggttgtcgg gacttagggt aggaggagta gaagcctgct 960

actgattctg tctctctctg tttctctctc ctccctccct ccctccttcc ctccctccct 1020

ccctcttctt cttcttctca tcctcctccc tcttcctctt cttcctccac ctccccaccc 1080

cttattttga tacagggttt ctctgtgtat ccctggctgt cctggaactc actctgtagc 1140

ccaggtgggc ctcgaactct cagcctcagt ccccagaatg ctgagaacac aggtctgagt 1200

gatcactgat ggctaaaagt tgggattaca ttgttgttgc ttgtttgttt attcttttgt 1260

acatgggacc caaatacaaa tagtagcctc aacaataaac acgggataag ttgctgctct 1320

gctttagggt ctccctgacc tctgtttttt tgttttttgt ttttggtatg ttttgttttc 1380

tgttgttgtt gttatcatgt ctataaatct atcttccttc ctccctcctt ccttcccttc 1440

ctacttccct ctctttcttc atccctccct tccccctact ctctttcacc cccagatagg 1500

aagcaagcat gataaaaacg tgtggtgttt tcctttttat gtagagagta ctgtgtagtg 1560

agtgttatcc tatgggtgct gccattctgc tgtatgttac ctgctgtatg ttataccaac 1620

ctagatggtg gtgaactcac atggttgctt ccatcttggt gaggttacca gccaagatgt 1680

ctgccctcca catgattgcc tccatcttgg tgaggtaaat gtttaataaa gtaacaaaac 1740

aagacattaa aaacaaacat tccagcacaa aatcttcttg agattagaga cataatagga 1800

agtcaggtga ggtcatacag gccattaatc ccatcacttg gggaacagag gcaggtagaa 1860

ctttgaattg aaggcaggta tatttagtga tttccagggc tatgtagaga ggcccctgac 1920

ccaactaata agtaaacagg aagataaata aatataatga acttagaata aaacaaagaa 1980

aggaagaaac aagggaaggc agtgctgggg gcctggctta tggtggatgg gggaattctg 2040

tgctagggtg cctgaaactc tgggctccat cctctgtagt gcataaactc tttggtacgt 2100

tagcccctgt ctgtaacaag gagctgtcca cggttgcagt actgcctttc ccatctcagc 2160

tgcccctcag gagctgtcca cagtggcgac actttttcat cctcagccta cagctttagg 2220

gaaacaccac tgcaggggct gtcccaaagg tgctgtccac agaggcagca ccttctctgt 2280

ggtctcaccc ctccagacac cccccagcag ccccacaggg atggcacctc agtaaaagcc 2340

aactgtggcc agagaagtct tcctacccta actcatagac tcgatgcagg gaaaacaggg 2400

tgaaaaaaag ccaccaagcc ctgagctccc cccagctcag gacttaaaat ctcatcaatc 2460

ctcactatgg aaatctctgc cttgagaagc tctgccccct cataaatcct atataagaac 2520

tgtccctttg tccagttccc tgccatccgc tcccaggagc agagggcagt tatccctgga 2580

ttcatccctc cacaccctgg acctgccaat aaacctttct tgagatttca tgcttcctgt 2640

gattctcagt ggaagaagcc aagaagaaaa gaaccaaaga gagggagcca ggctgaggct 2700

cctgagttct tcagctcagc tgtggatacc tgtgatggtt tgtatatgct ctgtccaggg 2760

aatggcatga ttagaaggtg tggccctgtt gaagtaggtg tgtcactgtg ggtgtgggct 2820

ataagaccct catcttaact gcatggaagt cattcttcca ctggcagcct tcagatgaaa 2880

atgtagaact ctcagctcct cctgcaccat gcctgcctag atgctgccct gctcccacct 2940

tgatgataat ggactgaacc tctgaacctg taagccagcc ccaattaaat gttgttcttt 3000

ataagtcttg ccttggttgt ggtgtctgtt cacagcagta aaaccatgac taagacaata 3060

tcttctactt ggagctgcaa caactctgct gaggaggctt cctctcagag ctatatggtt 3120

cctggtatct gtaaaattcc cttctattgg gacactttcc aacctcagat ctgtgtggtt 3180

cctggcccct gtgtctcggg atgcccttcc attagaacag cttctcccct cagagctgca 3240

tggttcctgg acttctgagt cccaggagac ccttccatct gagcagaaac acttacagga 3300

gcagagtcct ccaacaccac ggttttgttt tgaaagacca agaccaaccc tcaggaggtt 3360

tctggcaaag gcagattcta ggtgcacctg gaggagacct atagtgcagg accatccgtc 3420

gtaggttgct aggcaccaat gggcaaaggt agggaagaaa tcttaccaga agattctatt 3480

ccattccatt ctcctcacaa tgtaagagcc aaagttaacc tctaaggccc aagaacaagg 3540

taactctcca gaatgctggg agatgtagtt cttgggtaac aacaagccat gttctcgccc 3600

taaacaagtt tgtttgaatc aactacactg aatgtacttg atcatatgta ggagagagaa 3660

gattgattct agttcagggt ttcaggctat ttcagtccac catgatggga aaggcatgac 3720

attgtttatg acagtaagag catgtagcag aggatcctca catcacaaca ggccgaaatg 3780

cagaggacag tgcaaccaga ggacagtctg taactttcca agtccctctt ctagtgggtt 3840

gcttccacca cctctcaggt ggtgctacag ctcaggaaca attgagatgt gtgatgaagg 3900

gcaggtactc aactgtggct gtattgtatc cttttatagt tgtcctctgt gtgttgagct 3960

atgtgcgaga ttctcaggtc atcggagtac ctgttttact ttggcaggca taggagactc 4020

ctgagaactc tgcctgacat ccttgccagc ccaagctttg gtttagtgtg tgcagtatca 4080

ctcttgggtc ttatctgcat atccctgatg gcccatcaag atgtgt 4126

<210> 5

<211> 21

<212> DNA

<213> Mus musculus

<220>

<221> misc_

<223> SEQ ID NO 5-UTR sequence of mouse protein C exon 2

<400> 5

acaggtgtca gcagctccag g 21

<210> 6

<211> 104

<212> DNA

<213> Mus musculus

<220>

<221> misc_

<223> UTR sequence of exon 9 of SEQ ID NO 6-mouse protein C

<400> 6

cacccctccc tgctcacctc tggaccctag aagtcactct tggagtaagg ctgggctagt 60

gagtaccaag acagaggaca ttaaaggagc atgcaacaaa cata 104

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 7-LoxP site

<400> 7

ataacttcgt atagcataca ttatacgaag ttat 34

<210> 8

<211> 1386

<212> DNA

<213> Homo sapiens

<220>

<221> gene

<223> SEQ ID NO 8-human protein C coding sequence

<400> 8

atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60

gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120

aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180

gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240

gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300

tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360

gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420

tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480

agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540

ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600

acagaagacc aagaagacca agtagatccg cggctcattg atgggaagat gaccaggcgg 660

ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagctggc ctgcggggca 720

gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780

ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840

gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccaccga caatgacatc 900

gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960

ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020

ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080

aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140

gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200

gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260

agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320

tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380

ccttag 1386

<210> 9

<211> 13254

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 9-targeting vector from the 5 'end of the 5' homology arm to the 3 'end of the 3' homology arm

<400> 9

tccctgcaca gctagtcaca acgaaggaag gcgcttaggg aaccctggca gcttgcaaaa 60

cgcaaagggc tacggctgca tcgctctttt ccagacttct cagctgggag cttctggcag 120

ttttcccgag tcactccttt ctctcactag ctcacaaagt ggccagctga gtcagaagcc 180

tccttctagt acaggcctgc ctcccaccaa cgccatcaat caggacaagt aaggaagact 240

tctgagtcgc cccccccccc caccggtcaa atagagggga catcttatca ctgatggcat 300

cctagattgg tgatatatgt aattattttt gagtgtgcta cccacgaaca agctatatct 360

gtttatggtt gctgttgttt tggtttttgt tttcttttaa ggttctcatc cctcagccac 420

tgcgggcaaa aatgagacca catttgccaa taagtttgaa cacgctcaac cctctctttc 480

tccctccctt tctgatagac aattccttcg gtaggcagag gtgagcaatg ggcacacgga 540

gccttccaga gctgggatca gaaaacctct tgtttgtttg tctggggaga gggaggttcg 600

gcaccaaggg ctaagcaaat atttgcggtt atggattaac ctgactccca gactgacatg 660

gcgctacctg gacgaaattg cagtttctcc ttggcccacg cctgtaagtc cccctcattg 720

caagactgtg aaggactgtg gagggagggg aggggaggaa agtccagctg ggaggaaggt 780

gacgttcttg agctaaggct ctccaggcag actgaaatgt ggggccaagg aaaatgagcg 840

cccaaactct atctggacca aggcgtgggt tccctacaat ccaggtgacc atctcgacac 900

atgagatttt gtggatcaag tggacagcag tcaaagggtt ccctatgatc aggaaccatc 960

ctcagagcaa tcttgaaacc cagaacccca ctactcccct ctgccctgtt ctctaaggtg 1020

gactctacaa ttccggaagc cagcaaagcc gaagagtgag gccaggaggg gctgtgcagc 1080

tgggataggc gggcctgccc acctggtctg ggggacacta gaggccttgt ggtttgacta 1140

ctggttgggg gcaggggtta aggttccaga gttggggcca cataggcttg gccttgaagc 1200

caaactctgc cctcctctta ggtgtaggct tgtgacaagc cgcgtatctc ctccaagcct 1260

ttgggtccct tcccatgaaa tggaggtgag aatattcatg ccttcctctt ttaacagtga 1320

tcagtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1380

gtgtagagtg tggtttctgg tgttcagggt tgaacccaga atcttaaaca tgccaagtac 1440

gttctttcct actgaactgc aaccctccag tatcctgtac ttgttgtttg tttgtttgtt 1500

tgtttgtttg ttcggaagca cctgtggtgg cacacactta caatcctagt gctagagagc 1560

agaaacaagt ggatcccttg ggcttgctgg ctggccagcc tacgtgatga gtttcaggca 1620

gtgagagacc ttgtcccaaa caataaggtg gaagtaagcc atgatggcat acccctttag 1680

agctagtact cgagaggcag aagcaggtgg agtttaagac cagcctggtc tacatagaag 1740

ttccaggata gccaaaaagt acccaaggcc atccaaaaaa acaaaaacaa aacaaaacct 1800

ggagggaaaa aaaaaaccag acaatgcctg gggaaggatg aaggacagtc agtcagatta 1860

tccctggtca acacgtgtgc acaaatctgt gcacacaaga aagagcttca catgggttac 1920

tatttgtttt ccaacaactc atttttaagc ccccactctc tttctctgtt tttaaaaaag 1980

gtttatttat tttatgtata tgagtacatt attgctctct tcagacacac cagaaaagga 2040

cataagattc cattacagat ggttgtgagc caccatgtgg ttgctggaat ttgaactcag 2100

gtcctctgga agagcagtcg gtgctcttaa ccactgaacc atctccccag cccttccaac 2160

aactctttat ggaagaaacc tattctatcc attttataaa tgacagaact gaggcacgga 2220

gcacgtaaac atcttgttaa atacctctct ctctctctct ctctccccag taggaaatgg 2280

aatttgcccc aggcaatgac tttttttttt ttttttttgc tttcatgtac ctagagtaag 2340

cccagctcta aaggccacga gattgtctgt ctgtggaccg tggtgtaccc cactcccaga 2400

cccagcttcc acacagacaa tgagctcaca aacgtccttt actcccttcc ttccttgctt 2460

gctctgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttaaga gatcaacctc agacattttc 2520

tttaggagct atttaccttg ctttttgaga cagggcctcc ggatggcctg gagctagtca 2580

cctggagctg gtcaagcagg ctagggtggc tggcctaagc aatccacagg atctgcttat 2640

ctctgcctcc ccagtcctgg gattacaaga aacgcctcag acacctagat ttctgtttta 2700

attttctatg ggttctgggg atctttctta agtcttcaag tatgcacgga aagggcttta 2760

ttgactaaga tatctcccct gctcaggaat ggccctttca ttctactttg gagggagctg 2820

ggggtggggc ggggagcagc tcagctgtgt gtatccttgg agcttagaag ttctcctcag 2880

acaggtgtca gcagctccag gatgtggcag ctcacaagcc tcctgctgtt cgtggccacc 2940

tggggaattt ccggcacacc agctcctctt gactcagtgt tctccagcag cgagcgtgcc 3000

caccaggtgc tgcggatccg caaacgtgcc aactccttcc tggaggagct ccgtcacagc 3060

agcctggagc gggagtgcat agaggagatc tgtgacttcg aggaggccaa ggaaattttc 3120

caaaatgtgg atgacacact ggccttctgg tccaagcacg tcgacggtga ccagtgcttg 3180

gtcttgccct tggagcaccc gtgcgccagc ctgtgctgcg ggcacggcac gtgcatcgac 3240

ggcatcggca gcttcagctg cgactgccgc agcggctggg agggccgctt ctgccagcgc 3300

gaggtgagct tcctcaattg ctcgctggac aacggcggct gcacgcatta ctgcctagag 3360

gaggtgggct ggcggcgctg tagctgtgcg cctggctaca agctggggga cgacctcctg 3420

cagtgtcacc ccgcagtgaa gttcccttgt gggaggccct ggaagcggat ggagaagaag 3480

cgcagtcacc tgaaacgaga cacagaagac caagaagacc aagtagatcc gcggctcatt 3540

gatgggaaga tgaccaggcg gggagacagc ccctggcagg tggtcctgct ggactcaaag 3600

aagaagctgg cctgcggggc agtgctcatc cacccctcct gggtgctgac agcggcccac 3660

tgcatggatg agtccaagaa gctccttgtc aggcttggag agtatgacct gcggcgctgg 3720

gagaagtggg agctggacct ggacatcaag gaggtcttcg tccaccccaa ctacagcaag 3780

agcaccaccg acaatgacat cgcactgctg cacctggccc agcccgccac cctctcgcag 3840

accatagtgc ccatctgcct cccggacagc ggccttgcag agcgcgagct caatcaggcc 3900

ggccaggaga ccctcgtgac gggctggggc taccacagca gccgagagaa ggaggccaag 3960

agaaaccgca ccttcgtcct caacttcatc aagattcccg tggtcccgca caatgagtgc 4020

agcgaggtca tgagcaacat ggtgtctgag aacatgctgt gtgcgggcat cctcggggac 4080

cggcaggatg cctgcgaggg cgacagtggg gggcccatgg tcgcctcctt ccacggcacc 4140

tggttcctgg tgggcctggt gagctggggt gagggctgtg ggctccttca caactacggc 4200

gtttacacca aagtcagccg ctacctcgac tggatccatg ggcacatcag agacaaggaa 4260

gccccccaga agagctgggc accttagcac ccctccctgc tcacctctgg accctagaag 4320

tcactcttgg agtaaggctg ggctagtgag taccaagaca gaggacatta aaggagcatg 4380

caacaaacat acctccccga gtacctgtct gtcttttcat cctttttatg ggctattctg 4440

ggggaaagta acattaattg agcatgcact acacaccaag tctatgaaaa gaacctgctt 4500

aactcccaaa gcagttgtgt agaagatcta gtgggatctg agctgatatc acttctgggg 4560

gtgagtggag gagattgatt tagagaaagg aattttttta gaagttactg taagagacta 4620

atagagcctt tctcagggcc ttggaaagag cccgtgctag ttacatcaga aaagcttgcc 4680

agtgaccagt ggccagtgag actcagaatg gccatgtggt ggagccagga ttcaaaccaa 4740

ggtcacactc ccaaactcag ctgcttctct tctttattat ccctgggtgt gtgctggtgt 4800

gtgtgtgcgc gcgtgggtgt gtgggtggat acatgcatgt gtgtgtgtgt gtgtgtgtgt 4860

gtgtgtgtgt gtgtgtgtgt tatatgtttg gagaccagag gacaacttcg tttctcaaca 4920

ccatccactt gttttgtttt gtgttttgtt ttgtttgttg acacagggtc tctcactgtc 4980

ctgaaatcta cccagtaggc taggctggct ggctaccaaa ccccacccca ccctggcttt 5040

gacaagtgga gacagaagac cagtagtcca ctggagatgt gaccagatgc ccagaaggtg 5100

ctcctcatgg tgccctacag ttttgttgag gagtctgttt aataatgcag ctgggtgcag 5160

tggcagcacc tgtagccccc aatactgagg cagcattgct gcagtctgag aggtggggct 5220

cgagggacct aataacttcg tatagcatac attatacgaa gttatattaa gggttccgca 5280

agctctagtc gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc 5340

cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc 5400

ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga 5460

aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac 5520

agatggctgg caactagaag gcacagtcga ggctgatcag cgagctctag agaattgatc 5580

ccctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc 5640

gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc 5700

acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 5760

gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 5820

cacgacgaga tcatcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg 5880

cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 5940

agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 6000

aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 6060

gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 6120

ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 6180

ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 6240

aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 6300

ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 6360

tccatcttgt tcaatggccg atcccatggt ttagttcctc accttgtcgt attatactat 6420

gccgatatac tatgccgatg attaattgtc aacaggctgc aggtcgaaag gcccggagat 6480

gaggaagagg agaacagcgc ggcagacgtg cgcttttgaa gcgtgcagaa tgccgggcct 6540

ccggaggacc ttcgggcgcc cgccccgccc ctgagcccgc ccctgagccc gcccccggac 6600

ccaccccttc ccagcctctg agcccagaaa gcgaaggagc aaagctgcta ttggccgctg 6660

ccccaaaggc ctacccgctt ccattgctca gcggtgctgt ccatctgcac gagactagtg 6720

agacgtgcta cttccatttg tcacgtcctg cacgacgcga gctgcggggc gggggggaac 6780

ttcctgacta ggggaggagt agaaggtggc gcgaaggggc caccaaagaa cggagccggt 6840

tggcgcctac cggtggatgt ggaatgtgtg cgaggccaga ggccacttgt gtagcgccaa 6900

gtgcccagcg gggctgctaa agcgcatgct ccagactgcc ttgggaaaag cgcctcccct 6960

acccggtaga atttcgacga cctgcagcca agctagcttc gcgagctcga ccgaacaaac 7020

gacccaacac ccgtgcgttt tattctgtct ttttattgcc gctcagcttt acagtgacaa 7080

tgacggctgg cgactgaata ttagtgctta cagacagcac tacatatttt ccgtcgatgt 7140

tgaaatcctt tctcatatgt caccataaat atcaaataat tatagcaatc atttacgcgt 7200

taatggctaa tcgccatctt ccagcaggcg caccattgcc cctgtttcac tatccaggtt 7260

acggatatag ttcatgacaa tatttacatt ggtccagcca ccagcttgca tgatctccgg 7320

tattgaaact ccagcgcggg ccatatctcg cgcggctccg acacgggcac tgtgtccaga 7380

ccaggccagg tatctctgac cagagtcatc cttagcgccg taaatcaatc gatgagttgc 7440

ttcaaaaatc ccttccaggg cgcgagttga tagctggctg gtggcagatg gcgcggcaac 7500

accatttttt ctgacccggc aaaacaggta gttattcgga tcatcagcta caccagagac 7560

ggaaatccat cgctcgacca gtttagttac ccccaggcta agtgccttct ctacacctgc 7620

ggtgctaacc agcgttttcg ttctgccaat atggattaac attctcccac cgtcagtacg 7680

tgagatatct ttaaccctga tcctggcaat ttcggctata cgtaacaggg tgttataagc 7740

aatccccaga aatgccagat tacgtatatc ctggcagcga tcgctatttt ccatgagtga 7800

acgaacctgg tcgaaatcag tgcgttcgaa cgctagagcc tgttttgcac gttcaccggc 7860

atcaacgttt tcttttcgga tccgccgcat aaccagtgaa acagcattgc tgtcacttgg 7920

tcgtggcagc ccggaccgac gatgaagcat gtttagctgg cccaaatgtt gctggatagt 7980

ttttactgcc agaccgcgcg cctgaagata tagaagataa tcgcgaacat cttcaggttc 8040

tgcgggaaac catttccggt tattcaactt gcaccatgcc gcccacgacc ggcaaacgga 8100

cagaagcatt ttccaggtat gctcagaaaa cgcctggcga tccctgaaca tgtccatcag 8160

gttcttgcga acctcatcac tcgttgcatc gaccggtaat gcaggcaaat tttggtgtac 8220

ggtcagtaaa ttggacacct tcctcttctt cttgggcatg gccgcaggaa agcagagccc 8280

tgaagctccc atcaccggcc aataagagcc aagcctgcag tgtgacctca tagagcaatg 8340

tgccagccag cctgacccca agggccctca ggcttgggca cactgtctct aggaccctga 8400

gagaaagaca tacccatttc tgcttagggc cctgaggatg agcccagggg tggcttggca 8460

ctgaagcaaa ggacactggg gctcagctgg cagcaaagtg accaggatgc tgaggctttg 8520

acccagaagc cagaggccag aggccaggac ttctcttggt cccagtccac cctcactcag 8580

agctttacca atgccctctg gatagttgtc gggtaacggt ggacgccact gattctctgg 8640

ccagcctagg acttcgccat tccgctgatt ctgctcttcc agccactggc tgaccggttg 8700

gaagtactcc agcagtgcct tggcatccag ggcatctgag cctaccaggt ccttcagtac 8760

ctcctgccag ggcctggagc agccagcctg caacacctgc ctgccaagca gagtgaccac 8820

tgtgggcaca ggggacacag ggtggggccc acaacagcac cattgtccac ttgtccctca 8880

ctagtaaaag aactctaggg ttgcgggggg tgggggaggt ctctgtgagg ctggtaaggg 8940

atatttgcct ggcccatgga gctagcttgg ctggacgtaa actcctcttc agacctaata 9000

acttcgtata gcatacatta tacgaagtta tattaagggt tattgaatat gatcggaatt 9060

gggctgcagg aattcgatag cttggctgca ggtcgacgta cgtagcaagc ttgatgggcc 9120

ctggtaccac agcctgggca acacagcaaa aatcccttcc cttaaaaaaa acaaaagaga 9180

aggaagaagg acgaagtaga atgtggagga caaacagggg agagaggggg aaagaaaggg 9240

agggaattgt cttagagttt tacggctgtg cacagacacc atgatcaagg taactcttgt 9300

aaggataaca tttagttggg gctggcttac aggttcagaa gttcagtcca ttatcatcaa 9360

ggcaggaaca tggcagcatt caaggcagac atggtgcagg aggagctgag agttctacat 9420

cttcatctga agatttctag tagaatactg gcttccaggc agctaggatg agggtcttaa 9480

agcccacacc cagtgacaca cctactccaa cagggccaca cctcctaatc atgccactcc 9540

ctgggctgag catatagaaa ccatcacaga gtctaactag tgtggcccat cctgcaccca 9600

tggaagacca tcactggggc atagacaacc tccagagccc accctgacag ttcctgtctc 9660

tgccttctcc agcagtcacc agtttcaaat agctcctcaa ggacagatgg ggccttgtga 9720

gcttcacccc gctgcaggct ggaatgcgcc acctttaatc ccagcacttg gaaggcagag 9780

gcaggcagat ttctgagttc gaggccagcc tagtctacag agtgagttcc aggacagcca 9840

gggcgataca gagaaaccct gtctcaaaaa acaaaacaaa acaaaacgat agaaaagagc 9900

aaagtgacct tgggctatgg atgggatgga ccatcgggca ctgggttggg aagctgaact 9960

ggtccagatg cccagagccc agagctctct cctcagcagt tcataacctg gggtgttgcc 10020

acagcacaca cagcaaggtt agttctgctg gttgtcggga cttagggtag gaggagtaga 10080

agcctgctac tgattctgtc tctctctgtt tctctctcct ccctccctcc ctccttccct 10140

ccctccctcc ctcttcttct tcttctcatc ctcctccctc ttcctcttct tcctccacct 10200

ccccacccct tattttgata cagggtttct ctgtgtatcc ctggctgtcc tggaactcac 10260

tctgtagccc aggtgggcct cgaactctca gcctcagtcc ccagaatgct gagaacacag 10320

gtctgagtga tcactgatgg ctaaaagttg ggattacatt gttgttgctt gtttgtttat 10380

tcttttgtac atgggaccca aatacaaata gtagcctcaa caataaacac gggataagtt 10440

gctgctctgc tttagggtct ccctgacctc tgtttttttg ttttttgttt ttggtatgtt 10500

ttgttttctg ttgttgttgt tatcatgtct ataaatctat cttccttcct ccctccttcc 10560

ttcccttcct acttccctct ctttcttcat ccctcccttc cccctactct ctttcacccc 10620

cagataggaa gcaagcatga taaaaacgtg tggtgttttc ctttttatgt agagagtact 10680

gtgtagtgag tgttatccta tgggtgctgc cattctgctg tatgttacct gctgtatgtt 10740

ataccaacct agatggtggt gaactcacat ggttgcttcc atcttggtga ggttaccagc 10800

caagatgtct gccctccaca tgattgcctc catcttggtg aggtaaatgt ttaataaagt 10860

aacaaaacaa gacattaaaa acaaacattc cagcacaaaa tcttcttgag attagagaca 10920

taataggaag tcaggtgagg tcatacaggc cattaatccc atcacttggg gaacagaggc 10980

aggtagaact ttgaattgaa ggcaggtata tttagtgatt tccagggcta tgtagagagg 11040

cccctgaccc aactaataag taaacaggaa gataaataaa tataatgaac ttagaataaa 11100

acaaagaaag gaagaaacaa gggaaggcag tgctgggggc ctggcttatg gtggatgggg 11160

gaattctgtg ctagggtgcc tgaaactctg ggctccatcc tctgtagtgc ataaactctt 11220

tggtacgtta gcccctgtct gtaacaagga gctgtccacg gttgcagtac tgcctttccc 11280

atctcagctg cccctcagga gctgtccaca gtggcgacac tttttcatcc tcagcctaca 11340

gctttaggga aacaccactg caggggctgt cccaaaggtg ctgtccacag aggcagcacc 11400

ttctctgtgg tctcacccct ccagacaccc cccagcagcc ccacagggat ggcacctcag 11460

taaaagccaa ctgtggccag agaagtcttc ctaccctaac tcatagactc gatgcaggga 11520

aaacagggtg aaaaaaagcc accaagccct gagctccccc cagctcagga cttaaaatct 11580

catcaatcct cactatggaa atctctgcct tgagaagctc tgccccctca taaatcctat 11640

ataagaactg tccctttgtc cagttccctg ccatccgctc ccaggagcag agggcagtta 11700

tccctggatt catccctcca caccctggac ctgccaataa acctttcttg agatttcatg 11760

cttcctgtga ttctcagtgg aagaagccaa gaagaaaaga accaaagaga gggagccagg 11820

ctgaggctcc tgagttcttc agctcagctg tggatacctg tgatggtttg tatatgctct 11880

gtccagggaa tggcatgatt agaaggtgtg gccctgttga agtaggtgtg tcactgtggg 11940

tgtgggctat aagaccctca tcttaactgc atggaagtca ttcttccact ggcagccttc 12000

agatgaaaat gtagaactct cagctcctcc tgcaccatgc ctgcctagat gctgccctgc 12060

tcccaccttg atgataatgg actgaacctc tgaacctgta agccagcccc aattaaatgt 12120

tgttctttat aagtcttgcc ttggttgtgg tgtctgttca cagcagtaaa accatgacta 12180

agacaatatc ttctacttgg agctgcaaca actctgctga ggaggcttcc tctcagagct 12240

atatggttcc tggtatctgt aaaattccct tctattggga cactttccaa cctcagatct 12300

gtgtggttcc tggcccctgt gtctcgggat gcccttccat tagaacagct tctcccctca 12360

gagctgcatg gttcctggac ttctgagtcc caggagaccc ttccatctga gcagaaacac 12420

ttacaggagc agagtcctcc aacaccacgg ttttgttttg aaagaccaag accaaccctc 12480

aggaggtttc tggcaaaggc agattctagg tgcacctgga ggagacctat agtgcaggac 12540

catccgtcgt aggttgctag gcaccaatgg gcaaaggtag ggaagaaatc ttaccagaag 12600

attctattcc attccattct cctcacaatg taagagccaa agttaacctc taaggcccaa 12660

gaacaaggta actctccaga atgctgggag atgtagttct tgggtaacaa caagccatgt 12720

tctcgcccta aacaagtttg tttgaatcaa ctacactgaa tgtacttgat catatgtagg 12780

agagagaaga ttgattctag ttcagggttt caggctattt cagtccacca tgatgggaaa 12840

ggcatgacat tgtttatgac agtaagagca tgtagcagag gatcctcaca tcacaacagg 12900

ccgaaatgca gaggacagtg caaccagagg acagtctgta actttccaag tccctcttct 12960

agtgggttgc ttccaccacc tctcaggtgg tgctacagct caggaacaat tgagatgtgt 13020

gatgaagggc aggtactcaa ctgtggctgt attgtatcct tttatagttg tcctctgtgt 13080

gttgagctat gtgcgagatt ctcaggtcat cggagtacct gttttacttt ggcaggcata 13140

ggagactcct gagaactctg cctgacatcc ttgccagccc aagctttggt ttagtgtgtg 13200

cagtatcact cttgggtctt atctgcatat ccctgatggc ccatcaagat gtgt 13254

<210> 10

<211> 1386

<212> DNA

<213> Mus musculus

<220>

<221> gene

<223> SEQ ID NO 10-mouse protein C coding sequence

<400> 10

atgtggcaat tcagagtctt cctgctgctc atgtccacct ggggaatatc tagcataccg 60

gcccatcctg acccagtgtt ctccagcagc gagcatgccc accaggtgct tcgggtcaga 120

cgtgccaaca gcttcctgga agagatgcgg ccaggcagcc tggaacggga gtgtatggag 180

gagatctgtg acttcgagga ggcccaggag attttccaaa atgtggaaga cacactggcc 240

ttctggatca agtactttga cggtgaccag tgctcggctc cacccttgga ccaccagtgc 300

gacagcccat gctgcgggca tggcacttgc atcgacggca taggcagctt cagctgcagc 360

tgcgataagg gctgggaggg caagttctgt cagcaggagt tgcgcttcca ggactgtcgg 420

gtgaacaatg gcggctgctt gcactactgc ctggaggaga gcaatgggcg gcgctgcgct 480

tgtgccccgg gctatgagct ggcagacgac cacatgcgct gcaagtccac tgtgaatttt 540

ccatgtggga aactggggag gtggatagag aagaaacgca agatcctcaa acgagacaca 600

gacttagaag atgaactgga accagatcca aggatagtca acggaacgct gacgaagcag 660

ggtgacagtc cttggcaggc aatccttctg gactccaaga agaagctggc ctgcggaggg 720

gtgctcatcc acacttcctg ggtgctgacg gcagcccact gcgtggaggg caccaagaag 780

cttaccgtga ggcttggtga gtatgatctg cgacgcaggg accactggga gctggacctg 840

gacatcaagg agatcctcgt ccaccctaac tacacccgga gcagcagtga caacgacatt 900

gctctgctcc gcctagccca gccagccact ctctccaaaa ccatagtgcc catctgcctg 960

ccgaacaatg ggctcgctca gcaggagctc actcaggctg gccaggagac agtggtgaca 1020

ggctggggct atcaaagcga cagaatcaag gatggcagaa ggaaccgcac cttcatcctc 1080

accttcatcc gcatcccttt ggttgctcga aatgagtgcg tggaggtcat gaagaatgtg 1140

gtctcggaga acatgctgtg tgcaggcatc attgggaaca cgagagacgc ctgtgatggt 1200

gacagtgggg ggcccatggt ggtcttcttt cggggtacct ggttcctggt gggcctggtg 1260

agctggggtg agggctgtgg gcacaccaac aactatggca tctacaccaa agtgggaagc 1320

tacctcaaat ggattcacag ttacattggg gaaaagggtg tctcccttaa gagccagaag 1380

ctatag 1386

<210> 11

<211> 461

<212> PRT

<213> Homo sapiens

<220>

<221> PEPTIDE

<223> SEQ ID NO 11-human protein C amino acid sequence

<400> 11

Met Trp Gln Leu Thr Ser Leu Leu Leu Phe Val Ala Thr Trp Gly Ile

1 5 10 15

Ser Gly Thr Pro Ala Pro Leu Asp Ser Val Phe Ser Ser Ser Glu Arg

20 25 30

Ala His Gln Val Leu Arg Ile Arg Lys Arg Ala Asn Ser Phe Leu Glu

35 40 45

Glu Leu Arg His Ser Ser Leu Glu Arg Glu Cys Ile Glu Glu Ile Cys

50 55 60

Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln Asn Val Asp Asp Thr Leu

65 70 75 80

Ala Phe Trp Ser Lys His Val Asp Gly Asp Gln Cys Leu Val Leu Pro

85 90 95

Leu Glu His Pro Cys Ala Ser Leu Cys Cys Gly His Gly Thr Cys Ile

100 105 110

Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys Arg Ser Gly Trp Glu Gly

115 120 125

Arg Phe Cys Gln Arg Glu Val Ser Phe Leu Asn Cys Ser Leu Asp Asn

130 135 140

Gly Gly Cys Thr His Tyr Cys Leu Glu Glu Val Gly Trp Arg Arg Cys

145 150 155 160

Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp Asp Leu Leu Gln Cys His

165 170 175

Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Trp Lys Arg Met Glu Lys

180 185 190

Lys Arg Ser His Leu Lys Arg Asp Thr Glu Asp Gln Glu Asp Gln Val

195 200 205

Asp Pro Arg Leu Ile Asp Gly Lys Met Thr Arg Arg Gly Asp Ser Pro

210 215 220

Trp Gln Val Val Leu Leu Asp Ser Lys Lys Lys Leu Ala Cys Gly Ala

225 230 235 240

Val Leu Ile His Pro Ser Trp Val Leu Thr Ala Ala His Cys Met Asp

245 250 255

Glu Ser Lys Lys Leu Leu Val Arg Leu Gly Glu Tyr Asp Leu Arg Arg

260 265 270

Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile Lys Glu Val Phe Val His

275 280 285

Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn Asp Ile Ala Leu Leu His

290 295 300

Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr Ile Val Pro Ile Cys Leu

305 310 315 320

Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu Asn Gln Ala Gly Gln Glu

325 330 335

Thr Leu Val Thr Gly Trp Gly Tyr His Ser Ser Arg Glu Lys Glu Ala

340 345 350

Lys Arg Asn Arg Thr Phe Val Leu Asn Phe Ile Lys Ile Pro Val Val

355 360 365

Pro His Asn Glu Cys Ser Glu Val Met Ser Asn Met Val Ser Glu Asn

370 375 380

Met Leu Cys Ala Gly Ile Leu Gly Asp Arg Gln Asp Ala Cys Glu Gly

385 390 395 400

Asp Ser Gly Gly Pro Met Val Ala Ser Phe His Gly Thr Trp Phe Leu

405 410 415

Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gly Leu Leu His Asn Tyr

420 425 430

Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu Asp Trp Ile His Gly His

435 440 445

Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser Trp Ala Pro

450 455 460

<210> 12

<211> 461

<212> PRT

<213> Mus musculus

<220>

<221> PEPTIDE

<223> SEQ ID NO 12-mouse protein C amino acid sequence

<400> 12

Met Trp Gln Phe Arg Val Phe Leu Leu Leu Met Ser Thr Trp Gly Ile

1 5 10 15

Ser Ser Ile Pro Ala His Pro Asp Pro Val Phe Ser Ser Ser Glu His

20 25 30

Ala His Gln Val Leu Arg Val Arg Arg Ala Asn Ser Phe Leu Glu Glu

35 40 45

Met Arg Pro Gly Ser Leu Glu Arg Glu Cys Met Glu Glu Ile Cys Asp

50 55 60

Phe Glu Glu Ala Gln Glu Ile Phe Gln Asn Val Glu Asp Thr Leu Ala

65 70 75 80

Phe Trp Ile Lys Tyr Phe Asp Gly Asp Gln Cys Ser Ala Pro Pro Leu

85 90 95

Asp His Gln Cys Asp Ser Pro Cys Cys Gly His Gly Thr Cys Ile Asp

100 105 110

Gly Ile Gly Ser Phe Ser Cys Ser Cys Asp Lys Gly Trp Glu Gly Lys

115 120 125

Phe Cys Gln Gln Glu Leu Arg Phe Gln Asp Cys Arg Val Asn Asn Gly

130 135 140

Gly Cys Leu His Tyr Cys Leu Glu Glu Ser Asn Gly Arg Arg Cys Ala

145 150 155 160

Cys Ala Pro Gly Tyr Glu Leu Ala Asp Asp His Met Arg Cys Lys Ser

165 170 175

Thr Val Asn Phe Pro Cys Gly Lys Leu Gly Arg Trp Ile Glu Lys Lys

180 185 190

Arg Lys Ile Leu Lys Arg Asp Thr Asp Leu Glu Asp Glu Leu Glu Pro

195 200 205

Asp Pro Arg Ile Val Asn Gly Thr Leu Thr Lys Gln Gly Asp Ser Pro

210 215 220

Trp Gln Ala Ile Leu Leu Asp Ser Lys Lys Lys Leu Ala Cys Gly Gly

225 230 235 240

Val Leu Ile His Thr Ser Trp Val Leu Thr Ala Ala His Cys Val Glu

245 250 255

Gly Thr Lys Lys Leu Thr Val Arg Leu Gly Glu Tyr Asp Leu Arg Arg

260 265 270

Arg Asp His Trp Glu Leu Asp Leu Asp Ile Lys Glu Ile Leu Val His

275 280 285

Pro Asn Tyr Thr Arg Ser Ser Ser Asp Asn Asp Ile Ala Leu Leu Arg

290 295 300

Leu Ala Gln Pro Ala Thr Leu Ser Lys Thr Ile Val Pro Ile Cys Leu

305 310 315 320

Pro Asn Asn Gly Leu Ala Gln Gln Glu Leu Thr Gln Ala Gly Gln Glu

325 330 335

Thr Val Val Thr Gly Trp Gly Tyr Gln Ser Asp Arg Ile Lys Asp Gly

340 345 350

Arg Arg Asn Arg Thr Phe Ile Leu Thr Phe Ile Arg Ile Pro Leu Val

355 360 365

Ala Arg Asn Glu Cys Val Glu Val Met Lys Asn Val Val Ser Glu Asn

370 375 380

Met Leu Cys Ala Gly Ile Ile Gly Asn Thr Arg Asp Ala Cys Asp Gly

385 390 395 400

Asp Ser Gly Gly Pro Met Val Val Phe Phe Arg Gly Thr Trp Phe Leu

405 410 415

Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gly His Thr Asn Asn Tyr

420 425 430

Gly Ile Tyr Thr Lys Val Gly Ser Tyr Leu Lys Trp Ile His Ser Tyr

435 440 445

Ile Gly Glu Lys Gly Val Ser Leu Lys Ser Gln Lys Leu

450 455 460

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 13-Neo-F (P1)

<400> 13

aggctggtaa gggatatttg cctg 24

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 14-3' arm-R (P2)

<400> 14

gagtgagccc agacccataa caat 24

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 15-5' arm-F (P3)

<400> 15

tgggattaca agaaacgcct cagac 25

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 16-KI-R (P4)

<400> 16

aggagttggc acgtttgcgg at 22

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 17-KI-F (F1)

<400> 17

tgggattaca agaaacgcct cagac 25

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> 18-KI-R (R1) primer of SEQ ID NO

<400> 18

aggagttggc acgtttgcgg at 22

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 19-KI 2-F (F2)

<400> 19

ggctgtgggc tccttcacaa ctac 24

<210> 20

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 20-KI2-R (R2)

<400> 20

caggttcttt tcatagactt ggtgtgt 27

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primers of SEQ ID NO 21-Neo-del-F (F3)

<400> 21

agggacctaa taacttcgta tagc 24

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 22-Neo-del-R (R3)

<400> 22

cctgtttgtc ctccacattc tact 24

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 23-WT-F (F4)

<400> 23

catctacacc aaagtgggaa gc 22

<210> 24

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 24-KI2-R (R2)

<400> 24

caggttcttt tcatagactt ggtgtgt 27

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 25-SEQ-F (F1)

<400> 25

tgggattaca agaaacgcct cagac 25

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> primer of SEQ ID NO 26-SEQ-R (R3)

<400> 26

cctgtttgtc ctccacattc tact 24

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 27-hproC 1-F primer

<400> 27

tgggattaca agaaacgcct cagac 25

<210> 28

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 28-hproC 1-R primer

<400> 28

aggagttggc acgtttgcgg at 22

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 29-mproC-F primer

<400> 29

catctacacc aaagtgggaa gc 22

<210> 30

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 30-mproC-R primer

<400> 30

caggttcttt tcatagactt ggtgtgt 27

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 31-F8-common primer

<400> 31

gagcaaattc ctgtactgac 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 32-F8-WT-forward primer

<400> 32

tgcaaggcct gggcttattt 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_

<223> SEQ ID NO 33-F8-Mut-forward primer

<400> 33

tgtgtcccgc cccttccttt 20

Claims

1. A method for modifying the gene of mouse animal model features that at least one copy of the endogenous nucleotide sequence for coding protein C in the genome of mouse animal model is substituted by the nucleotide sequence for coding human protein C, or the functional fragment of coding human protein C, or the functional mutant of coding human protein C.

2. A method of genetically modifying a mouse animal model according to claim 1, wherein both copies of the endogenous nucleotide sequence encoding protein C in the genome of the mouse animal model are replaced by a nucleotide sequence encoding human protein C or encoding a functional fragment or mutant of human protein C.

3. A method of genetically modifying a mouse animal model according to claim 1 or 2, wherein the strain of mouse is laboratory mouse C57 BL/6.

4. A genetically modified mouse animal model genetic modification method according to claim 1 or 2, wherein the endogenous nucleotide sequence encoding protein C in the genome of the mouse has been replaced with a nucleotide sequence encoding human protein C.

5. A method of genetically modifying a mouse animal model according to any one of claims 1 or 2, wherein the nucleotide sequence encoding human protein C comprises SEQ ID NO:8, or a nucleotide sequence identical to SEQ ID NO:8 with at least 80% sequence identity.

6. A method of genetically modifying a mouse animal model according to any one of claims 1 or 2, wherein the expression of the nucleotide sequence encoding human protein C or a functional fragment or mutant encoding human protein C is regulated by endogenous regulatory sequences of the protein C gene of the mouse animal model.

7. A method of genetically modifying a mouse animal model according to any one of claims 1 or 2, wherein the start codon of the nucleotide sequence encoding human protein C or a functional fragment or mutant of human protein C is located in the protein C gene of the genome of the mouse animal model at a position corresponding to the start codon of the nucleotide sequence encoding endogenous protein C of the mouse animal model, and the stop codon of the nucleotide sequence encoding human protein C or a functional fragment or mutant of human protein C is located in the genome of the mouse animal model at a position corresponding to the stop codon of the nucleotide sequence encoding endogenous protein C of the mouse animal model.

8. A method of genetically modifying a mouse animal model according to any one of claims 1 or 2, wherein the animal comprises in its genome one or more genetic modifications which down-regulate or inactivate a gene encoding coagulation factor VIII and/or a gene encoding coagulation factor IX.

9. A genetically modified mouse animal model genetic modification method according to any one of claims 1 or 2, wherein the animal has knocked out a gene encoding coagulation factor VIII and/or a gene encoding coagulation factor IX.

10. A method of producing a genetically modified mouse animal model according to any one of claims 1 to 9, characterized in that the method comprises:

(i) providing a mouse pluripotent stem cell wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the mouse pluripotent stem cell has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant of human protein C; and

(ii) generating a genetically modified mouse animal model from the mouse pluripotent stem cells.

11. The method of claim 10, wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the mouse pluripotent stem cell has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant of human protein C.

12. The method according to claim 10 or 11, characterized in that the mouse pluripotent stem cells are mouse embryonic stem cells.

13. A vector for homologous recombination in a mouse pluripotent stem cell, wherein the vector is capable of replacing at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the mouse pluripotent stem cell with a nucleotide sequence encoding human protein C or a functional fragment or mutant encoding human protein C.

14. A vector for use in homologous recombination in mouse pluripotent stem cells according to claim 13, wherein the vector functionally comprises: (i) a nucleotide sequence encoding human protein C or encoding a functional fragment of human protein C or encoding a functional mutant of human protein C; and (ii) at least one marker for positive selection; (iii)5' -homology arm; (iv)3' -homologous arm.

15. A vector for use in homologous recombination in a mouse pluripotent stem cell according to claim 13, wherein the vector comprises in 5' to 3' order (i) a 5' -homology arm, (ii) a nucleotide sequence encoding human protein C or a nucleotide sequence encoding a functional fragment of human protein C or a nucleotide sequence encoding a functional mutant of human protein C; (iii) a first portion of a 3' homology arm; (iv) a first recombination site for a specific site; (v) a marker for positive selection; (vi) (vii) a second site-specific recombination site, and (vii) a second portion of the 3' homology arm.

16. The vector according to claim 13, wherein said vector comprises, in order from 5 'to 3': (i) comprises the amino acid sequence of SEQ ID NO:2, 5' -homology arm of the nucleotide sequence of seq id no; and (ii) a nucleotide sequence encoding human protein C; (iii) a first portion of a 3' -homology arm, the first portion comprising SEQ ID NO: 3; (iv) a first loxP recombination site comprising SEQ ID NO: 7; (v) a positive selection marker for the neomycin resistance gene; (vi) a second loxP recombination site comprising SEQ ID NO: 7; (vii) a second portion of the 3' -homology arm, the second portion comprising SEQ ID NO: 4.

17. A vector according to any one of claims 13 to 16 for use in homologous recombination in a mouse pluripotent stem cell, wherein the vector comprises SEQ ID NO: 9.

18. A vector according to any one of claims 13 to 16 for use in homologous recombination in a mouse pluripotent stem cell, wherein the vector comprises SEQ ID NO: 1.

19. A method of producing a mouse pluripotent stem cell wherein at least one copy of an endogenous nucleotide sequence encoding protein C in the genome of the mouse pluripotent stem cell has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or a functional mutant of human protein C, the method comprising:

(i) transfecting a mouse pluripotent stem cell with a vector for homologous recombination in a mouse pluripotent stem cell according to any one of claims 13 to 18; and

(ii) (ii) selecting the one or more transfected mouse pluripotent stem cells of (i) to identify one or more mouse pluripotent stem cell clones whose at least one copy of an endogenous nucleotide sequence encoding protein C in the genome has been replaced with a nucleotide sequence encoding human protein C or a functional fragment or mutant of human protein C.

20. The method of claim 19, wherein the mouse pluripotent stem cell is a mouse embryonic stem cell.

21. Use of the genetically modified mouse animal model of any one of claims 1 to 9 for testing a candidate therapeutic agent.