Genetically Modified Non-Human Animals

Abstract

The present invention relates generally to genetically modified non-human animals. The present invention relates to a genetically modified non-human animal, in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human animal has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C. The invention also relates to vectors, cells and methods for the production of such non-human animals. The invention also relates to methods of testing agents for their ability to alter to the level and/or functional activity of human protein C and thus provides methods of testing agents for their potential therapeutic efficacy.

Claims

1. A genetically modified non-human animal, in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human animal has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C.

2. The genetically modified non-human animal of claim 1, in which both copies of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human animal have been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C.

3. The genetically modified non-human animal of claim 1, wherein said animal is a mouse.

4. The genetically modified non-human animal of claim 1, wherein said endogenous nucleotide sequence encoding Protein C in the genome of said non-human animal has been replaced by a nucleotide sequence encoding human Protein C.

5. The genetically modified non-human animal of claim 1, wherein said nucleotide sequence encoding human Protein C comprises the nucleotide sequence of SEQ ID NO:8, or a sequence having at least 80% sequence identity to SEQ ID NO:8.

6. The genetically modified non-human animal of claim 1, wherein expression of said nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C is under the control of the endogenous regulatory sequences of the Protein C gene of the non-human animal.

7. The genetically modified non-human animal of claim 1, wherein the start codon of the nucleotide sequence encoding human Protein C (or functional fragment or functional variant thereof) is positioned in the Protein C gene in the genome of the non-human animal at the position corresponding to the start codon of the nucleotide sequence encoding endogenous Protein C, and the stop codon of the nucleotide sequence encoding human Protein C (or functional fragment or functional variant thereof) is positioned in the Protein C gene in the genome of the non-human animal at the position corresponding to the stop codon of the nucleotide sequence encoding endogenous Protein C.

8. The genetically modified non-human animal of claim 1, wherein said animal comprises one or more genetic modifications in its genome that down-regulate or inactivate or knock-out one or more other genes, preferably said one or more other genes encode a blood clotting factor, preferably Factor VIII or Factor IX.

9. The genetically modified non-human animal of claim 1, wherein a gene encoding Factor VIII is knocked-out and/or a gene encoding Factor IX is knocked out.

10. A cell, or cell line, derived from a genetically modified non-human animal of claim 1, in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said cell or cell line has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C.

11. A non-human pluripotent stem cell, preferably a non-human embryonic stem cell, in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human pluripotent stem cell has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C.

12. The non-human pluripotent stem cell of claim 11, wherein said non-human pluripotent stem cell is a mouse pluripotent stem cell.

13. A vector for homologous recombination in a non-human pluripotent stem cell, wherein said vector is capable of replacing at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human pluripotent stem cell with a nucleotide sequence encoding human Protein C encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C.

14. The vector of claim 13, said vector comprising, (a) in functional combination, (i) a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C; (ii) at least one marker for positive selection; (iii) a 5′-homology arm; and (iv) a 3′-homology arm; or (b) in order from 5′ to 3′, (i) a 5′-homology arm, (ii) a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C (preferably a nucleotide sequence encoding human Protein C); (iii) a first part of a 3′-homology arm; (iv) a first site-specific recombination site; (v) a marker for positive selection; (vi) a second site-specific recombination site and (vii) a second part of a 3′-homology arm; or (c) in order from 5′ to 3′, (i) a 5′-homology arm comprising a nucleotide sequence of SEQ ID NO:2; (ii) a nucleotide sequence encoding human Protein C; (iii) a first part of a 3′-homology arm said first part comprising a nucleotide sequence of SEQ ID NO:3; (iv) a first loxP recombination site comprising a nucleotide sequence of SEQ ID NO:7; (v) a marker for positive selection that is the neomycin resistance gene; (vi) a second loxP recombination site comprising a nucleotide sequence of SEQ ID NO:7; and (vii) a second part of a 3′-homology arm said second part comprising a nucleotide sequence of SEQ ID NO:4; or (d) a nucleotide sequence of SEQ ID NO:9; or (e) a nucleotide sequence of SEQ ID NO:1.

15. A method for producing a non-human pluripotent stem cell, preferably a non-human embryonic stem cell, in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human pluripotent stem cell has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C, said method comprising: transfecting non-human pluripotent stem cells with a vector of claim 13; and (ii) selecting one or more transfected non-human pluripotent stem cells of (i) to identify one or more non-human pluripotent stem cell clones in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human pluripotent stem cell has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C.

16. The method of claim 15, wherein said non-human pluripotent stem cell is a mouse pluripotent stem cell.

17. A method of producing a genetically modified non-human animal of claim 1, said method comprising (i) providing a non-human pluripotent stem cell in which at least one copy of the endogenous nucleotide sequence encoding Protein C in the genome of said non-human pluripotent stem cell has been replaced by a nucleotide sequence encoding human Protein C, encoding a functional fragment of human Protein C or encoding a functional variant of human Protein C; and (ii) generating a genetically modified non-human animal from said non-human pluripotent stem cell.

18. A non-human pluripotent stem cell produced by the method of claim 15.

19. A method of testing one or more agents to identify the potential use of the one or more agents in therapy, said method comprising (a) providing a genetically modified non-human animal of claim 1; and (b) administering to said animal one or more agents to be tested.

20. (canceled)

21. A genetically modified non-human animal produced by the method of claim 17.

Description

[0215] The invention will now be further described in the following non-limiting Example with reference to the following drawings.

[0216] FIG. 1: Schematic depiction of the wildtype mouse Protein C allele (A), the targeting vector (B), the targeted allele (C), and the constitutive knock-in (KI) allele (after Ned deletion) (D). The elements labelled 1, 2 and 9 are exons. A 5′ UTR (untranslated region) is part of exon 2 of mouse Protein C and a 3′ UTR is part of exon 9 of mouse Protein C. These UTRs are present in the targeting vector and in the targeted allele. Exon 1 consists only of UTR. Human PROC CDS is the coding sequence of human protein C. Neo.sup.r=Neo selection cassette. DTA=diphtheria toxin A negative selection marker. Although not depicted in FIG. 1A, the wildtype mouse Protein C allele also comprises exons 3, 4, 5, 6, 7 and 8 (and intervening introns) between exons 2 and 9.

[0217] FIG. 2: Further schematic depiction of the targeting vector.

[0218] FIG. 3: The targeting vector was digested with the indicated restriction enzymes and the resultant digest products were separated by gel electrophoresis. The left-hand image shows the results of digestion with the indicated restriction enzymes (M: Marker; 1: ApaL1; 2: AhdI; 3: FspI/HindIII; 4: NcoI; 5: SacI; 6: NotI). The nucleic acid fragment sizes (in kilo-base pairs (kb)) with the various restriction enzyme digests are indicated.

[0219] FIG. 4: Schematic depiction of the PCR screening strategy for screening in homologous recombination of the targeting construct in mouse embryonic stem (ES) cells. P1, P2, P3 and P4 are oligonucleotide primers used for the PCR screening. Human PROC CDS is the coding sequence of human protein C. Ned=Neo selection cassette.

[0220] FIG. 5: Gel electrophoresis of PCR products obtained following the 3′ arm PCR using primers P1 and P2 as depicted in FIG. 4. M: Marker. An expanded view of the Marker is shown in the left hand panels. WT: Wildtype. The various identifiers (e.g. 2H7, etc.) are the identifiers of the ES clones being screened.

[0221] FIG. 6: Gel electrophoresis of PCR products obtained following the KI PCR using primers P3 and P4 as depicted in FIG. 4. M: Marker. An expanded view of the Marker is shown in the left hand panels. WT: Wildtype. The various identifiers (e.g. 2H7, etc.) are the identifiers of the ES clones being screened.

[0222] FIG. 7: Schematic depiction of the Southern blot strategy for screening correct targeting of the construct in mouse embryonic stem (ES) cells. The elements labelled 1, 2 and 9 are exons. A 5′ UTR (untranslated region) is part of exon 2 of mouse Protein C and a 3′ UTR is part of exon 9 of mouse Protein C. These UTRs are present in the targeting vector and in the targeted allele. Exon 1 consists only of UTR. Human PROC CDS is the coding sequence of human protein C. Neo.sup.r=Neo selection cassette (also known as the neomycin resistance gene). EcoNI and Bsu36I are restriction enzymes. Although not depicted in FIG. 7A, the wildtype mouse Protein C allele also comprises exons 3, 4, 5, 6, 7 and 8 (and intervening introns) between exons 2 and 9.

[0223] FIG. 8: Results of Southern blot analysis following digestion of genomic DNA from the ES cell clones with the indicated restriction enzymes, electrophoresis of the digestion products and probing with a probe which hybridises to the Ned cassette. WT: Wildtype. The various identifiers (e.g. 2H7, etc.) are the identifiers of the ES clones being screened.

[0224] FIG. 9: Schematic depiction of the mouse genotyping strategy. F1, F2, F3, F4, R1, R2 and R3 are oligonucleotide primers used for this PCR-based genotyping screening. The elements labelled 1, 2 and 9 are exons. A 5′ UTR (untranslated region) is part of exon 2 of mouse Protein C and a 3′ UTR is part of exon 9 of mouse Protein C. Exon 1 consists only of UTR. Human PROC CDS is the coding sequence of human protein C. Neo.sup.r=Neo selection cassette. Although not depicted in FIG. 9A, the wildtype mouse Protein C allele also comprises exons 3, 4, 5, 6, 7 and 8 (and intervening introns) between exons 2 and 9.

[0225] FIG. 10: (A) Gel electrophoresis of PCR products obtained following the KI1 PCR using primers F1 and R1 as depicted in FIG. 9. (B) Gel electrophoresis of PCR products obtained following the KI2 PCR using primers F2 and R2 as depicted in FIG. 9. (C) Gel electrophoresis of PCR products obtained following the Neo deletion PCR using primers F3, R3, F4 and R2 as depicted in FIG. 9. M: Marker. An expanded view of the Marker is provided. ESC: Embryonic Stem Cell. WT: Wildtype. MT: mutant allele (i.e. constitutive knock-in (KI) allele after Neo deletion). The lanes numbered 1-7 correspond to samples obtained from pups 1 #-7 #.

[0226] FIG. 11: Gel electrophoresis of PCR products obtained following PCR using primers F1 and R3 as depicted in FIG. 9. M: Marker. An expanded view of the Marker is provided. WT: Wildtype. MT: mutant allele (i.e. constitutive knock-in (KI) allele after Neo deletion).

[0227] FIG. 12: Graphs showing bleeding times in mouse tail clipping experiments. (A) Hemophilia A model. (B) Hemophilia B model. n=number of mice. WT=Wildtype. F8−/−(control)=F8−/− mice without Factor VIII administration. F8−/−(FVIII)=F8−/− mice with Factor VIII administration. Hproc+/+F8−/−(control)=Hproc+/+F8−/− mice without Factor VIII administration. Hproc+/+F8−/−(FVIII): Hproc+/+F8−/− mice with Factor VIII administration. F9−/−(control)=F9−/− mice without Factor IX administration. F9−/−(FIX)=F9−/− mice with Factor IX administration. Hproc+/+F9−/−(control)=Hproc+/+F9−/− mice without Factor IX administration. Hproc+/+F9−/−(FIX): Hproc+/+F9−/− mice with Factor IX administration.

[0228] FIG. 13: Schematic depiction of clotting cascade. TF=Tissue Factor. fVII=factor VII. fVIIa=activated factor VII. fX=factor X. fXa=activated factor X. fVa=activated factor V. fIX=factor IX. fIXa=activated factor IX. fXIa=activated factor XI. fVIIIa=activated factor VIII. fXIII=factor XIII. fXIIIa=activated factor XIII APC: Activated 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, available at: creativecommons.org/licenses/by/4.0/.)

EXAMPLE

Human Protein C Constitutive Knock-in Mouse Model

Targeting Vector

[0229] A targeting vector for generating a knock-in of human protein C into the mouse (C57BL/6) protein C locus was constructed. The nucleotide sequence of the targeting vector is set forth in SEQ ID NO:1.

[0230] The mouse protein C gene (NCBI Reference Sequence: NM_001042767.3) is located on mouse chromosome 18. Nine exons have been identified, with the ATG start codon in exon 2 and TAG stop codon in exon 9.

[0231] The human protein C gene (NCBI Reference Sequence: NM_000312.3) is located on human chromosome 2. Nine exons have been identified, with the ATG start codon in exon 2 and TAG stop codon in exon 9.

[0232] For the knock-in model mouse described herein, the region from ATG start codon to TAG stop codon of mouse protein C was replaced with the coding sequence of human protein C. Thus, the targeting vector includes the coding sequence of human protein C flanked by homology arms for targeting to the mouse protein C locus to achieve targeted replacement of the nucleotide sequence encoding mouse protein C with the human protein C coding sequence.

[0233] The targeting vector further comprises, positioned 3′ with respect to the human protein C coding sequence (human PROC CDS), a Neo cassette (Ned) flanked by loxP sites. Expression of the Neo cassette can be selected for using the antibiotic G418. The targeting vector further comprises, positioned outside of the homology arms and positioned 5′ with respect to the 5′ end of the 5′ homology arm, a DTA (diphtheria toxin A) negative selection marker.

[0234] Mouse genomic fragments containing homology arms (HAs) were amplified from BAC clone by using high fidelity Taq DNA polymerase, and were sequentially assembled into a targeting vector together with site-specific (loxP) recombination sites and selection markers, and the human Protein C coding sequence.

[0235] A schematic depiction of the wildtype mouse Protein C allele, the targeting vector, the targeted allele, and the constitutive knock-in (KI) allele (after Neo′ deletion) is set forth in FIG. 1.

[0236] A further depiction of the targeting vector is provided in FIG. 2.

[0237] The targeting vector was digested by restriction enzymes for confirmation purposes. The results of these confirmatory restriction enzyme digests are shown in FIG. 3. These results demonstrate that the targeting vector was correctly constructed.

[0238] Correct construction of the targeting vector was also confirmed by nucleic acid sequencing.

Generation of Human Protein C Constitutive Knock-in Mouse Embryonic Stem (ES) Cells

[0239] The human protein C targeting construct (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/6 ES cells according to standard electroporation procedures. The transfected ES cells were subject to G418 selection (200 μg/mL) 24 hours post electroporation. 564 G418 resistant clones were picked and amplified in 96-well plates. Two copies of 96-well plates were made, one copy was frozen down and stored at −80° C. and the other copy of the 96-well plates was used for DNA isolation and subsequence PCR screening for homologous recombination. The PCR screening identified 16 potential targeted clones, from among which 12 were expanded and further characterized by Southern blot analysis. Eight of the twelve expanded clones were confirmed to be correctly targeted. The PCR screening and Southern blot analysis is described in more detail below.

[0240] The regions shown in FIG. 4 were selected for PCR screening. The PCR screening was performed as follows:

3′ Arm PCR

Primers for 3′Arm PCR:

[0241]

TABLE-US-00003 Neo-F (P1): (SEQ ID NO: 13) 5′-AGGCTGGTAAGGGATATTTGCCTG-3′ 3′arm-R (P2): (SEQ ID NO: 14) 5′-GAGTGAGCCCAGACCCATAACAAT-3′

Expected PCR Product:

Wildtype: None

Targeted: ˜4.5 kb

Reaction Mix:

[0242]

TABLE-US-00004 Component x1 ES cell genomic DNA 2.0 μl Forward primer (10 μM) 0.8 μl Reverse primer (10 μM) 0.8 μl dNTPs (2.5 mM) 2.4 μl 5X LongAmp Taq Reaction 4.0 μl LongAmp Taq DNA 1.2 μl Polymerase ddH.sub.2O 8.8 μl Total 20.0 μl 

Cycling Condition:

[0243]

TABLE-US-00005 Step Temp. Time Cycles Initial denaturation 94° C. 3 min Denaturation 94° C. 30 s 33 x Annealing 60° C. 30 s Extension 65° C. 50 s/kb Additional extension 65° C. 10 min
The results of the 3′ arm PCR screening are shown in FIG. 5.
The potentially targeted clones were further screened by PCR for the presence of the knock-in (KI) site.

Primers for KI PCR:

[0244]

TABLE-US-00006 5′arm-F (P3): (SEQ ID NO: 15) 5′-TGGGATTACAAGAAACGCCTCAGAC-3′ KI-R (P4): (SEQ ID NO: 16) 5′-AGGAGTTGGCACGTTTGCGGAT-3′

Expected PCR Product:

Wildtype: None

Targeted: 380 bp

Reaction Mix:

[0245]

TABLE-US-00007 Component x1 ES cell genomic DNA 1.5 μl Forward primer (10 μM) 1.0 μl Reverse primer (10 μM) 1.0 μl P112 Taq DNA Polymerase 12.5 μl  ddH.sub.2O 9.0 μl Total 25.0 μl 

Cycling Condition:

[0246]

TABLE-US-00008 Step Temp. Time Cycles Initial denaturation 94° C. 3 min Denaturation 94° C. 30 s 33 x Annealing 60° C. 30 s Extension 72° C. 30 s Additional extension 72° C. 5 min Storage temperature 25° C.

[0247] The results of the KI PCR screening are shown in FIG. 6.

[0248] Based on the PCR screening, samples 2H7, 3B2, 3E2, 3C4, 3D8, 3C11, 4B2, 4A4, 4H3, 4G10, 4A12, 5C5, 5G6, 5G8, 6H3 and 6B5 were shown as potentially targeted ES clones.

[0249] Positive clones (2H7, 4A12, 4B2, 3C4, 3C11, 5G6, 4A4, 3B2, 6B5, 3D8, 5G8 and 4G10) from PCR screening were expanded and further characterized by Southern blot analysis. The Southern strategy is shown in FIG. 7. The genomic DNA was digested with either Bsu36I or EcoNI, and hybridized using a Neo probe. The Neo probe is expected to detect the following DNA fragment from targeted allele in the Southern analysis: ˜10.37 kb (with Bsu36I digestion) and ˜11.39 kb (with EcoNI digestion).

[0250] Expected Fragment Sizes for Southern Blot:

[0251] Neo Probe (containing 5′ arm)—10.37 kb-Bsu36I

[0252] Neo Probe (containing 3′ arm)—11.39 kb-EcoNI

[0253] Eight of the twelve clones (2H7, 4B2, 3C4, 3C11, 4A4, 3D8, 5G8 and 4G10) were confirmed to be correctly targeted by Southern blot analysis. The results of the Southern blot analysis are shown in FIG. 8.

Generation of Human Protein C Constitutive Knock-in Mice

[0254] Targeted ES cell clone 3C4 was injected into C57BL/6 albino embryos, which were then re-implanted into CD-1 pseudo-pregnant females. Founder animals were identified by their coat color, their germline transmission was confirmed by breeding with C57BL/6 females and subsequent genotyping of the offspring. Cre mouse (i.e. a Cre recombinase expressing mouse) was used to mate with F0 (founder animals) to generate F1 mice in which the Neo′ cassette that was flanked by loxP sites was deleted. Four male and two female heterozygous targeted mice were generated from clone 3C4. Further details of the mouse genotyping strategy are provided below:

[0255] The regions shown in FIG. 9 were selected for PCR-based genotyping of the mice.

K11 PCR

Primers for KI1 PCR:

[0256]

TABLE-US-00009 KI-F (F1): (SEQ ID NO: 17) 5′-TGGGATTACAAGAAACGCCTCAGAC-3′ KI-R (R1): (SEQ ID NO: 18) 5′-AGGAGTTGGCACGTTTGCGGAT-3′

Expected PCR Product:

Wildtype: N.A.

Targeted: 380 bp

Reaction Mix:

[0257]

TABLE-US-00010 Component x1 Mouse genomic DNA 1.5 μl Forward primer (10 μM) 1.0 μl Reverse primer (10 μM) 1.0 μl Premix Taq Polymerase 12.5 μl ddH.sub.2O 9.0 μl Total 25.0 μl

Cycling Condition:

[0258]

TABLE-US-00011 Step Temp. Time Cycles Initial denaturation 94° C. 3 min Denaturation 94° C. 30 s 33 x Annealing 62° C. 35 s Extension 72° C. 35 s Additional extension 72° C. 5 min

K12 PCR

Primers for KI2 PCR:

[0259]

TABLE-US-00012 KI2-F (F2): (SEQ ID NO: 19) 5′-GGCTGTGGGCTCCTTCACAACTAC-3′ KI2-R (R2): (SEQ ID NO: 20) 5′-CAGGTTCTTTTCATAGACTTGGTGTGT-3′

Expected PCR Product:

Wildtype: N.A.

Targeted: 324 bp

[0260] Reaction Mix:

TABLE-US-00013 Component x1 Mouse genomic DNA 1.5 μl Forward primer (10 μM) 1.0 μl Reverse primer (10 μM) 1.0 μl Premix Taq Polymerase 12.5 μl  ddH.sub.2O 9.0 μl Total 25.0 μl 

Cycling Condition:

[0261]

TABLE-US-00014 Step Temp. Time Cycles Initial denaturation 94° C. 3 min Denaturation 94° C. 30 s 33 x Annealing 62° C. 35 s Extension 72° C. 35 s Additional extension 72° C. 5 min

Neo Deletion PCR

Primers for Neo Deletion PCR:

[0262]

TABLE-US-00015 Neo-del-F (F3): (SEQ ID NO: 21) 5′-AGGGACCTAATAACTTCGTATAGC-3′ Neo-del-R (R3): (SEQ ID NO: 22) 5′-CCTGTTTGTCCTCCACATTCTACT-3′ WT-F (F4): (SEQ ID NO: 23) 5′-CATCTACACCAAAGTGGGAAGC-3′ KI2-R (R2): (SEQ ID NO: 24) 5′-CAGGTTCTTTTCATAGACTTGGTGTGT-3′

Expected PCR Product:

Wildtype: 298 bp

Targeted: 230 bp

Reaction Mix:

[0263]

TABLE-US-00016 Component x1 Mouse genomic DNA 1.5 μl Forward primer1 (F3) (10 μM) 1.0 μl Reverse primer1 (R3) (10 μM) 1.0 μl Forward primer2 (F4) (10 μM) 0.5 μl Reverse primer2 (R2) (10 μM) 0.5 μl Premix Taq Polymerase 12.5 μl  ddH.sub.2O 8.0 μl Total 25.0 μl 

Cycling Condition:

[0264]

TABLE-US-00017 Step Temp. Time Cycles Initial denaturation 94° C. 3 min Denaturation 94° C. 30 s 33 x Annealing 62° C. 35 s Extension 72° C. 35 s Additional extension 72° C. 5 min

Results of the Mouse Genotyping PCRs:

[0265] Seven pups (1 #, 2 #, 3 #, 4 #, 5 #, 6 # and 7 #) from clone 3C4 were identified positive (i.e. positive for the presence of the constitutive human protein C knock-in (KI) allele) by PCR screening for KI1, KI2 and Neo deletion as described above, the positive pups were reconfirmed by PCR screening for Neo deletion. The PCR results for the Neo deletion PCR confirmed that these pups were heterozygous for the constitutive human protein C knock-in (KI) allele. The Neo deletion PCR also confirms that the 3C4 ES cells were heterozygous for the targeted allele. Note: one mouse (6 #) died. The results of the mouse PCR-based genotyping are shown in FIG. 10.
PCR amplification from mouse DNA was also done using primers F1 and R3 and the PCR amplified fragment (which includes the coding sequence (CDS) of human Protein C and also mouse UTR sequence) was sequenced and no mutations were found. An image of the PCR amplified fragments run on an electrophoretic gel is presented in FIG. 11.

[0266] Primers for Sequencing PCR:

TABLE-US-00018 Seq-F (F1): (SEQ ID NO: 25) 5′-TGGGATTACAAGAAACGCCTCAGAC-3′ Seq-R (R3): (SEQ ID NO: 26) 5′-CCTGTTTGTCCTCCACATTCTACT-3′

[0267] Expected PCR Product:

[0268] Wildtype: N.A.

[0269] Product Size: 2795 bp

Reaction Mix:

[0270]

TABLE-US-00019 Component x1 DNA 2.0 μL Forward primer (10 μM) 0.8 μL Reverse primer (10 μM) 0.8 μL dNTPs (2.5 mM) 2.4 μL 5X LongAmp Taq Reaction 4.0 μL Long Amp Taq DNA Polymerase 1.2 μL ddH.sub.2O 8.8 μL Total 20.0 μL 

Cycling Condition:

[0271]

TABLE-US-00020 Step Temp. Time Cycles Initial denaturation 94° C. 3 min Denaturation 94° C. 30 s 33 x Annealing 60° C. 30 s Extension 65° C. 50 s/kb Additional extension 65° C. 10 min

Generating Homozygous Human Protein C Knock-in Mice and Generation of Further Mice Having the Human Protein C Knock-in and Also Factor VIII Deficiency and Generation of Further Mice Having the Human Protein C Knock-in and Also Factor IX Deficiency

[0272] A. Heterozygous human protein C (hproC+/−) mice mated and 58 offspring mice were genotyped by PCR using the following primers:

TABLE-US-00021 hproC 1-F: (SEQ ID NO: 27) 5′-TGGGATTACAAGAAACGCCTCAGAC-3′ hproC 1-R: (SEQ ID NO: 28) 5′-AGGAGTTGGCACGTTTGCGGAT-3′
Expected PCR Product: Wildtype: None, Targeted: 380 bp. This hproC primer pair recognizes the human protein C knock-in allele but does not recognize the wildtype (i.e. non-targeted) mouse protein C allele.

TABLE-US-00022 mproC-F: (SEQ ID NO: 29) 5′-CATCTACACCAAAGTGGGAAGC-3′ mpro-R: (SEQ ID NO: 30) 5′-CAGGTTCTTTTCATAGACTTGGTGTGT-3′
Expected PCR Product: 280 bp from wildtype mice and heterozygous human protein C mice. This mproC primer pair can detect the mouse protein C allele (but not the human protein C targeted allele) and thus can detect wildtype mice, heterozygous human protein C knock-in mice, but not homozygous human protein C knock-in mice.

TABLE-US-00023 Genotype Mice quantity Theory yield Actual yield hproC+/+ 3 25% 5.2% hproC+/− 39 50% 67.2% hproC−/− 16 25% 27.6%

[0273] For the avoidance of doubt, the genotype hproC+/+ means that the mouse is homozygous for the human Protein C coding sequence (i.e. homozygous for the human Protein C “knock-in” allele). As the targeting vector results in the replacement of the mouse Protein C coding sequence with the human Protein C coding sequence it thus follows that mice of the genotype hproC+/+ do not comprise the mouse Protein C coding sequence. The genotype hproC+/− means that the mouse is heterozygous for the human Protein C coding sequence (i.e. heterozygous for the human Protein C “knock-in”). Thus, a mouse with the genotype hproC+/− comprises the coding sequence for human Protein C (human Protein C “knock-in”) and the coding sequence for mouse Protein C. The genotype hproC−/− means that the mouse is homozygous for mouse Protein C and does not comprise the human Protein C coding sequence (i.e. does not comprise the human Protein C “knock-in”). [0274] B. Male heterozygous human protein C (hproC+/−) mice mated with female factor VIII deficient mice (F8−/−; the F8−/− mice are completely devoid of factor VIII; F8−/− mice are from Jackson Laboratory, US) and 37 offspring mice were genotyped by PCR using the following primers for F8 genotyping (and also the mproC and hproC primers described above in part A):

TABLE-US-00024 F8-Common: (SEQ ID NO: 31) 5′-GAG CAA ATT CCT GTA CTG AC-3′ F8-WT-Forward: (SEQ ID NO: 32) 5′-TGC AAG GCC TGG GCT TAT TT-3′ F8-Mut-Forward: (SEQ ID NO: 33) 5′-TGT GTC CCG CCC CTT CCT TT-3′

Expected PCR Product: WT (+) F8=620 bp, Mutant (−) F8=420 bp

[0275]

TABLE-US-00025 Genotype Mice quantity Theory yield Actual yield hproC+/−, F8+/− 18 50% 48.6% hproC−/−, F8+/− 19 50% 51.4%

[0276] For the avoidance of doubt, the F8-WT-Forward and F8-Mut-Forward primers discriminate between the wildtype Factor VIII (F8) allele and the mutant Factor VIII (F8) allele. [0277] C. Male heterozygous human protein C and heterozygous factor VIII (hproC+/−, F8+/−) mice mated with female factor VIII deficient mice (F8−/−) and 31 offspring mice were genotyped by PCR. Note: The F8 (factor VIII) gene is located on X chromosome in mouse. Therefore male heterozygous F8 deficient mice do not have wildtype F8 gene and are equivalent to homozygous F8 deficient mice for mating purposes.

TABLE-US-00026 Genotype Mice quantity Theory yield Actual yield hproC+/−, F8−/− 17 50% 54.8% hproC−/−, F8−/− 14 50% 45.2% [0278] D. Heterozygous human protein C mice (hproC+/−) mated with heterozygous human protein C and heterozygous factor VIII mice (hproC+/−, F8+/−) and 12 offspring mice were genotyped by PCR

TABLE-US-00027 Genotype Mice quantity Theory yield Actual yield hproC+/+, F8+/− 2 25% 16.7% hproC+/−, F8+/−; 5 50% 41.7% hproC+/−, F8+/+ hproC−/−, F8+/−; 5 25% 41.7% hproC−/−, F8+/+ [0279] E. Male homozygous human protein C and heterozygous factor VIII mice (hproC+/+, F8+/−) mated with female heterozygous human protein C and factor VIII deficient mice (hproC+/−, F8−/−) and 25 offspring mice were genotyped by PCR

TABLE-US-00028 Genotype Mice quantity Expected yield Actual yield hproC+/+, F8−/− 12 50% 48% hproC+/−, F8−/− 13 50% 52% [0280] F. Male heterozygous human protein C (hproC+/−) mice mated with female factor IX deficient mice (F9+/−; the F9+/− mice are partially devoid of factor IX; F9+/− mice are from Jackson Laboratory, US) and 10 offspring mice were genotyped by PCR using the following primers for F9 genotyping (and also the mproC and hproC primers described above in part A):

TABLE-US-00029 F9-Common: (SEQ ID NO: 34) 5′-AAC AGG GAT AGT AAG ATT GTT CC-3′ F9-WT: (SEQ ID NO: 35) 5′-TGG AAG CAG TAT GTT GGT AA GC-3′ F9-Mut: (SEQ ID NO: 36) 5′-TCC TGT CAT CTC ACC TTG CTC-3′

Expected PCR Product: WT(+)F9=620 bp, Mutant(−)F9=420 bp

[0281]

TABLE-US-00030 Genotype Mice quantity Theory yield Actual yield hproC+/−, F9+/+ 5 50% 50% hproC+/−, F9+/− 2 25% 20% hproC+/−, F9−/− 3 25% 30%
For the avoidance of doubt, the F9-WT and F9-Mut primers discriminate between the wildtype Factor IX (F9) allele and the mutant Factor IX (F9) allele. Note: The F9 (factor IX) gene is located on X chromosome in mouse. Therefore, male heterozygous F9 deficient mice do not have wildtype F9 gene and are equivalent to homozygous F9 deficient mice for mating purposes. [0282] G. Male heterozygous human protein C and factor IX deficient mice (hproC+/−, F9+/−) mated with female heterozygous factor IX mice (F9+/−) and 5 offspring mice were genotyped by PCR.

TABLE-US-00031 Genotype Mice quantity Theory yield Actual yield hproC+/−, F9+/+ 0 12.5% 0 hproC+/−, F9+/− 0 .sup. 25% 0 hproC+/−, F9−/− 1 12.5% 20% hproC−/−, F9+/+ 1 12.5% 20% hproC−/−, F9+/− 1 .sup. 25% 20% hproC−/−, F9−/− 2 12.5% 40% [0283] H. Male heterozygous human protein C and factor IX deficient mice (hproC+/−, F9+/−) mated with female heterozygous human protein C and heterozygous factor IX mice (hproC+/−, F9+/−) and 21 offspring mice were genotyped by PCR.

TABLE-US-00032 Genotype Mice quantity Theory yield Actual yield hproC+/−, F9+/+ 3 12.5% 14.3% hproC+/−, F9+/− 4 12.5% .sup. 19% hproC+/−, F9−/− 2 .sup. 25%  9.5% hproC−/−, F9+/+ 2 6.75%  9.5% hproC−/−, F9+/− 0 6.75% 0 hproC−/−, F9−/− 3 12.5% 14.3% hproC+/+, F9+/+ 2 6.75%  9.5% hproC+/+, F9+/− 1 6.75% 4.75% hproC+/+, F9−/− 4 12.5% .sup. 19% [0284] I. Male heterozygous human protein C and factor IX deficient mice (hproC+/−, F9+/−) mated with female heterozygous human protein C and factor IX deficient mice (hproC+/−, F9−/−) and 4 offspring mice were genotyped by PCR

TABLE-US-00033 Genotype Mice quantity Theory yield Actual yield hproC−/−, F9−/− 1 25% 25% hproC+/−, F9−/− 1 50% 25% hproC+/+, F9−/− 2 25% 50%

Hemophilia Mouse Models

[0285] For hemophilia A model, 8 to 10-week-old mice including WT mice, factor VIII deficient mice (F8−/−) and human protein C knockin and factor VIII deficient double mutant mice (hproC+/+, F8−/−) anesthetized using 80 mg/kg sodium pentobarbital i.p. were placed on their abdomen with the tail immersed in 37° C. saline. The distal tail of mice was transected at 4 mm (severe injury), and the bleeding was arterial and venous. Human factor VIII was injected into an orbital vein 5 min before the tail was transected. Controls were also performed in which no Human Factor VIII was injected. For hemophilia B model, the same procedures were conducted except that factor IX deficient mice (F9−/−), human protein C knockin and factor IX deficient double mutant mice (hproC+/+, F9−/−), and human factor IX were used. Bleeding time was measured following the tail-tip transection and immediate immersion of the tail in 10 ml of saline at 37° C. Bleeding time was set at cessation of blood leakage for at least 1 min. After 15 min, the tail was removed from the saline and the bleeding time measure was ended.

RESULTS AND DISCUSSION

[0286] Humanized protein C knockin mice were generated by targeted inactivation of the murine protein C gene with a human protein C expression cassette, as described above. The mice (hproC+/+) are viable and able to cross with mouse disease models such as coagulation factor VIII deficient (F8−/−) or factor IX deficient (F9−/−) mice and such models are useful to study the functions of human protein C and activated protein C in mice in vivo.

[0287] Human protein C knockin and factor VIII deficient (hproC+/+, F8−/−) or humanized protein C knockin and factor IX deficient (hproC+/+, F9−/−) double mutant mice were generated as described above. These mice appear to be normal and healthy without challenges. If the double mutant hproC+/+F8−/− or double mutant hproC+/+F9−/− mice are challenged by tail cut bleeding, the prolonged bleeding time is comparable to the factor VIII deficient mice (F8−/−) or factor IX deficient mice (F9−/−), respectively. Prolonged bleeding time is the characteristic symptom of hemophilia. Human factor VIII or human factor IX could correct the prolonged bleeding time in factor VIII deficient mice or factor IX deficient mice, respectively, as well as in the respective double mutant mice. The results of the bleeding time study are shown in FIG. 12.

[0288] That the bleeding characteristics observed with the double mutant mice (hproC+/+, F8−/−) or (hproC+/+, F9−/−) which express human Protein C (but not mouse protein C) are very consistent with the bleeding characteristics observed with the respective factor VIII deficient mice (F8−/−) or factor IX deficient mice (F9−/−) which express endogenous mouse Protein C (but do not contain the human Protein C knock-in) is desirable and advantageous as this indicates that the human Protein C can functionally complement for the mouse Protein C that has been removed in the double mutant mice. These results indicate that mice having the human Protein C knock-in will be useful models for studying haemophilia or other pathophysiological conditions involving protein C pathway. The double mutant models described in this example (hproC+/+, F8−/−; and hproC+/+, F9−/−) are such models. These models are invaluable and represent the first mouse model for in vivo testing therapeutic candidate agents targeting human protein C or APC.

[0289] The double mutant mouse model (hproC+/+, F8−/−) described herein is useful as a model for testing potential candidate therapeutic agents targeting human protein C or APC. This double mutant mouse model is particularly useful as Factor VIII deficiency is characteristic of haemophilia A (classic haemophilia) and thus these mice provide a model of the situation in haemophilia A subjects. Without wishing to be bound by theory, blood clotting in Factor VIII deficient subjects (e.g. haemophilia A sufferers) can still occur, albeit at a much slower rate. Although Factor VIII is a major target of APC, in a Factor VIII deficient subject APC can still exert anticoagulant activity via its inhibitory effect on activated Factor V (fVa). Activated Factor V is essential for clotting and is a major target of APC. Without wishing to be bound by theory, targeting (inhibiting) human Protein C/APC in a Factor VIII deficient animal (e.g. a haemophilia sufferer characterised by a Factor VIII deficiency) can improve clotting by reducing the inhibition of activated Factor V (fVa). FIG. 13 provides a schematic diagram of the clotting cascade.

[0290] The double mutant mouse model (hproC+/+, F9−/−) described herein is useful as a model for testing potential candidate therapeutic agents targeting human protein C or APC. This double mutant mouse model is particularly useful as Factor IX deficiency is characteristic of haemophilia B and thus these mice provide a model of the situation in haemophilia B subjects.

[0291] Other hproC+/+ knock-in mice of the invention (i.e. other than the hproC+/+F8−/− or hproC+/+F9−/− double mutants described in the present Example) would also be useful mouse models for studying human Protein C (or human activated Protein C, APC) in vivo and identifying potential Protein C/APC targeting therapeutic agents. For example, hproC+/+ knock-in mice that are also deficient in Factor X or Factor XI would represent a useful mouse model as it would represent the situation in human patients who are deficient in Factor X or Factor XI.

[0292] In the context of Factor VIII and/or Factor IX deficiency or absence (which characterise certain haemophilias), factor X could still be activated by tissue factor (TF) and activated Factor VII (fVIIa). In the presence of activated factor X (fXa) and activated Factor V (fVa) (which as mentioned above is essential for clotting), prothrombin could be activated into thrombin to initiate clotting. Without wishing to be bound by theory, targeting (inhibiting) human Protein C/APC, e.g. in Factor VIII and/or Factor IX deficient animals, could improve clotting by reducing the inhibition of activated Factor V (fVa).

[0293] hproC+/+ knock-in mice that do not contain any further genetic modifications (e.g. no knockouts of other genes) would also be useful mouse models for studying human Protein C/APC activity in vivo and testing potential candidate therapeutic agents targeting human protein C or APC, not only with a view to identifying potential haemophilia therapies, but also therapeutic agents useful in other pathophysiological conditions involving the protein C pathway.