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Definitions

• vCJD in contrast to the other human prion diseases, the disease- associated form of the prion protein and infectivity is readily detectable in lymphoid tissues throughout the body (Hill, A. F. et al., Lancet 353: 183-9 (1999); Head, M. W. et al., Am. J. Pathol. 164: 142 (2004)), even before the onset of clinical disease (Hilton, D. A. et al., Lancet 352:703-4 (1998)). This gives rise to concerns that blood and blood products may also contain infectious particles, representing a possible source of iatrogenic spread of variant CJD.
• the risk of transmitting prion-related disorders through human products is a serious health concern.
• the method of prevention is on one hand the handling of vCJD donators combined with f. e. clearing the blood by filtration.
• human proteins preventing the risk of prion transmission through blood transfusion by contaminated donors.
• organisms such as Escherichia coli and Saccharomyces cerevisiae
• human proteins require additional post-translational modifications to perform the function of the endogenous protein and thus require their synthesis in mammalian cells or even species-specific cell lines for the plasma-like functioning of the produced protein.
• non-human cell lines such as Chinese hamster ovary cells (CHO)
• CHO Chinese hamster ovary cells
• the purified therapeutic proteins are contaminated with cellular trace components causing antigenic reactions in the patients (Refacto, Wyeth, package insert).
• proteins expressed by non-human expression systems may have non-human glycosylation patterns also giving rise to antigenic reactions in the patient.
• Biological stability and efficacy of clotting factors is substantially influenced by their O-and N-glycosylation pattern. Especially peripheral and terminal monosaccharides are important, because they are detected by specific receptors from cells which are responsible for their degradation. Clotting fac- tors carry sialic acid residues as terminal monosaccharides. Modification in the composition of sialic acids in the antennae of glycoproteins can result in heterogeneous glycosylation patterns. Thus, biological stability and efficacy are crucially affected when modification occurs.
• mammalian, especially human, systems are preferable for the production of recombinant human proteins.
• the immortalized cell line HEK293 and its derivates, f. e. Freestyle HEK293F are capable of expressing recombinant human proteins (EP 05 105 965.7). Since many therapeutics are produced in mammalian systems, there is a need for ensuring the safety of these products isolated from such systems regarding complete absence of prion proteins.
• Such a knockout cell line production system as the one described in this invention completely prevents expression of the prion protein and will provide re- combinant human proteins absolutely free from infectious prion proteins and free of a risk of prion contamination for the patients, who receive recombinant pharmaceuticals produced in these prion-free cells.
• RNAi interference RNA
• knockout technologies have been mostly developed in mouse ES cells (Joyner A. L., Oxford Univ. Press, UK 1993).
• ES murine embryonic stem
• Today, the knockout technology in the mouse has developed far enough to allow not only complete knockout ("null alleles"), but also condi- tional mutants.
• the targeted mutation may be temporally induced or sup- pressed, e.g. by supplementing the mouse chow with tetracycline (tet-on, tet- off systems, Gossen, M, and Bujard f H. Proc. Natl. Acad. ScL USA 89: 5547- 5551 (1992)); it may be driven by a cell- or tissue-specific promoter, or by an ubiquitous but developmentally regulated promoter which may be shut off during embryonic development but quickly activated after birth.
• the generation of a knockout ES cell line always requires the same initial steps: (1) generation of a targeting construct specifically designed to knock out a particular gene; (2) introduction of this construct in the ES cells and (3) selection and screening of cells bearing a targeted deletion of the desired gene.
• the targeting construct must always contain a selection cassette and a significant region of homology to the gene which will be knocked out. These two elements are absolutely necessary to achieve and detect integration only in the desired locus: the region of homology to the endogenous gene triggers homologous recombination between the two homologous DNA fragments, whereas the selection cassette allows screening of cells bearing the integrated construct.
• neomycin phosphotransferase cassette consisting of the ORF encoding this enzyme driven by a mouse phosphoglycerate kinase (pGK) pro- moter (Soriano et al., Cell 64, 693-702 (1991)) and flanked downstream by a polyA transcription termination signal.
• pGK mouse phosphoglycerate kinase
• the frequency with which the targeting construct integrates in the desired region of the genome is much lower than that of random integration, which makes it usually necessary to screen several hundreds of antibi- otic-resistant clones before a correctly targeted one is identified.
• One possibility to increase the targeting frequency of a given construct is to increase the length of the region of homology, since there is a linear relationship between both parameters (Hasty et al., MoI Cell Biol. 1991 Nov; 11(11) : 5586-91 (1991)).
• a number of "trap-approaches” have been developed which rely on the use of elements of the target locus for efficient expression of the selection cassette.
• the targeting vector is designed in such a way that the transcriptional machinery of the endogenous target gene will drive expression of the selection cassette cloned in the targeting vector.
• the vector con- tains homologous regions to the target gene which do not have any promoter activity, and therefore most of the clones in which the integration has occurred at random cannot survive antibiotic selection.
• promoter trap selections achieve enrichments of 100-fold for targeted clones.
• PrP gene has been so far only transiently and partially silenced in scrapie-infected, mouse neuroblastoma cells in culture (Daude et al., J. Cell. ScL. 2003 JuI l; 116(Pt 13) : 2775-9 (2003)).
• mice homozygous for the inactivated gene i.e., fully devoid of PrP expression
• mice homozygous for the inactivated gene are indeed resistant to prion infection.
• Most remarkably however, all of the above mentioned PrP knockout mice are viable and developmentally normal. Only a relatively mild neurological phenotype has been characterized in the Nagasaki, RcmO and Zurich II knockouts (Rossi et al., EMBO J. 20, 4, 694-702, (2001)), indicating that the function of the PrP gene is not absolutely essential.
• knockout technology has encountered two major problems when applied to somatic cells.
• knocking out a gene in these cells only provides limited information about the functional consequences of the disruption, since the phenotype of knockout cells in culture does not necessarily reflect the final effect in a mutant organism such as the mouse derived from knockout ES cells.
• the second, and more critical problem is that the targeting frequency of homologous recombination in somatic cells is about two orders of magnitude lower than in ES cells (Hanson and Sedivy, MoI. Cell Biol. 15(1) :45-51 (1995)).
• promoter trap approaches utilising promoterless vectors are absolutely essential (Sedivy and Dutriaux, Trends Genet.
• the selection cassette can only be expressed after homologous recombination from the promoter of the target gene.
• Gene targeting can also be improved by the ClonePix technology available from Genetix, UK.
• a somatic human cell line e.g. a cell line derived from the HEK293F cell line
• the gene coding for the prion protein sequence has been inactivated in at least one, preferably in both alleles or three alleles in triploid cells.
• Such resulting prion- ablated cells are suitable for recombinant production of human target proteins after transfection with a suitable expression vector encoding the target protein.
• this method provides an effective system to produce safe and highly active recombinant human proteins free from prion proteins for therapeutic applica- tion in humans.
• the present invention relates to
• a prion protein(PrP)-free, immortalized, somatic, cell line wherein both alleles of the PrP gene were completely deleted (2) a method for producing the PrP free immortalized cell line as defined in (1) above, which comprises subsequent deletion of both alleles of the PrP ORF in a respective starting cell by homologous recombination with PrP knock-out constructs; (3) a PrP knock-out construct as defined in (2) above; (4) the use of the PrP-free immortalized cell line as defined in (1) above for PrP-free recombinant production of a human protein or a derivative or mutant thereof (hereinafter "target protein");
• a method for preparing a cell line for PrP-free recombinant production of a target protein which comprises transfecting a PrP-free immortalized host cell line as defined in (1) above with a transfection vector comprising an origin of replication, and a gene encoding said human target protein, whereby the gene for the human target protein is linked at its 5' end with a promoter and its 3' end with a polyA signal; (6) a PrP-free immortalized cell line stably transfected, preferably under serum-free conditions, with the transfection vector as defined in (5) above; and
• a method for PrP-free recombinant production of a human target protein to be used as pharmaceuticals which comprises culturing a PrP-free, immortalized, human cell line as defined in (6) above.
• the method of embodiment (7) of the invention is particularly suitable for the production of recombinant human proteins and therapeutical antibodies, including clotting factors like factor VII/a, factor VIII, factor IX, von Willebrand factor (vWF) and Adamtsl3 and growth factors like granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF), free from prion protein contamination.
• an immortalized human cell line e.g. a HEK 293 cell line, with an ablated prion protein sequence of the invention is utilized.
• This cell line is obtainable by transfecting an immortalized human cell line with a vector containing a selectable or selection marker such as for example the neomycin phosphotransferase ORF, devoid of its own promoter and translation initiation.
• the cell line is a human cell line, like a cell line derived from HEK 293F or Per.C ⁇ cells (immortalized human foetal Retina cells).
• HEK 293F HEK 293F
• Per.C ⁇ cells immortalized human foetal Retina cells.
• Other suitable cells are CHO (Chinese Hamster Ovary cells) and BHK (Baby Hamster Kidney cells) cells.
• Figure 1 Promoter trap strategy to ablate the human PrP gene
• Figure 2 1.
• Figure 3 Cloning strategy to generate construct pBS_Neo_P " _L+R_Arm_2B.
• FIG. 6 Genomic PCR screening after integration of targeting construct pBS_Neo_P-_R+L_Arm_2B.
• DNA marker GeneRuler DNA Ladder Mix
• Positive control genomic DNA from a targeted PrP cell mix population.
• Al-8 and Bl-8 are the cell clones picked by Clone- PixFL. The clones labelled with green circle were identified as PCR positive clones due to the 2,3 kb bands.
• Figure 7 Southern analysis of PrP KO. cells after targeted integration of the first K.O. construct pBS_Neo_P-_R+L_Arm_2B: the following is utilized : DNA marker: GeneRuler DNA Ladder Mix; WT: 293F wild type: K.O. : a K.O. clone identified after targeted integration of the 1st K.O. construct pBS_Neo_P-_R+L_Arm_2B. Genomic DNA from K.O. clone and 293F WT cells was hybridized with the 5'-, 3'- and Neo-probes.
• DNA marker GeneRuler DNA Ladder Mix
• WT 293F wild type: K.O. : a K.O. clone identified after targeted integration of the 1st K.O. construct pBS_Neo_P-_R+L_Arm_2B.
• Genomic DNA from K.O. clone and 293F WT cells was hybridized with
• WT 293F cells show a 10,8 kb WT band with with both 5'- and 3'-probes but no signal with the Neo-probe.
• a 4,2 kb band with 3'-probe and a 6,5 kb band with 3'-probe could be detected, additionally a 6,5 kb band was seen with the Neo-probe.
• Figure 8 FISH analysis of PrP K.O. cells with one targeted PrP allele: pattern a: two signals of chr20 and Bac; Pattern b: 3 signals chr20 and Bac; pattern c: 3 signals of chr20 but only 2 signals of Bac.
• Figure 9 ELISA analysis of PrP K.O. cells with one targeted PrP allele and 293F cells: 2 K.O. cell lines bearing one PrP targeted allele (K.O. 1 and K.O. 6) were analyzed and compared to wild type 293F cells.
• PrP K.O. integrations In the gel the following is utilized : DNA marker: GeneRuler DNA Ladder Mix; Positive control : genomic DNA
• T3-1 is a mixed cell population selected with zeocin.
• T3-2 is mixed cell population se- lected with zeocin and G418. Both mixed cell populations were identified as positive.
• Figure 12 Genomic Southern strategy to characterize clones after stable integration of the targeting construct pBS_Neo-_P-_L+R_Arm_.
• Fig. 13 Transfection efficiency of PrP KO cells with one PrP targeted allele. The same amount of cells were transfected with a expression plasmid and transfection efficiency was compared.
• Fig. 14 Expression of FVIII (Fig. 14a), FIX (Fig. 14b) and G-CSFb (Fig. 14c) in PrP K. O. cells with one targeted PrP allele. Expression of arbitrary units of FVIII, FIX and G-CSFb per 10E6 cells compared to 293 F wild type cells.
• BAC means bacterial artificial chromosome
• bp means base pairs.
• G418 and zeocin are two different selection antibiotics; stably transfected cells with constructs carrying them as selection markers become resistant (“G418 R “ or “zeocin R ”) to these antibiotics.
• Homologous recombination refers to a mechanism whereby two DNA fragments of homologous sequence recombine with each other.
• Left arm refers to the intronic region of the PrP gene located immediately upstream of exon3.
• Light arm refers to the region immediately downstream of exon3 of the PrP gene.
• Neo refers to the neomycin phosphotransferase gene.
• ORF means open reading frame.
• PCR means polymerase chain reaction
• PrP means prion gene or the prion protein.
• HEK293F refers to a specific human embryonic kidney cell line.
• embodiment (1) of the invention pertains to a prion protein (PrP)-free, immortalized, somatic, human cell line wherein both alleles of the PrP gene have been completely deleted.
• said cell line is capable of being transfected and being cultured under serum-free conditions.
• the cell line has been rendered immortal by integration of adenoviral sequences into its genome.
• the cell line may be derived from a starting cell selected from the group of kidney, bladder, liver, lung, cardiac muscle, smooth muscle, ovary and gastrointestinal cells.
• the starting cell is a human kidney cell line, such as a human foetal kidney cell.
• the foetal human kidney cell is either a Freestyle 293 (293F cells; Invitrogen R79007), a HEK 293 (293 cells; ATCC CRL-1573; DSM ACC 305), or a 293T CeII(DSM ACC 2494), preferably is a 293F cell (Invitrogen R79007).
• the PrP ORF has been completely deleted by homologous recombination with knockout traps carrying selectable or selection marker genes so that expression of the selectable or selection maker is driven by the endogenous PrP promoter.
• the knock-out constructs may be suitable to delete the entire PrP ORF in both alleles. Further, the knock-out constructs may carry the same or different promoterless selection marker genes flanked by two sequences homologous to the insertion site within the PrP gene of the starting cell. It is, however, preferred that the knockout constructs carry different selection marker genes or selectable markers.
• the knock-out constructs may further carry one of the following functional sequences: a poly A sequence, recombinase recognition sequences, IRES and the like.
• the homologous sequences of the knock-out construct may have a length of 1 to 10 kb, preferably of about 6 kb and do preferably correspond to sequences upstream and downstream of the PrP ORF of the starting cell line. Particularly preferred is that the homologous sequences are those shown in SEQ ID NOs:2 and 3.
• Suitable selection markers encode positive selection markers including, but not limited to, neomycin phosphotransferase, zeocin, hygromycin; and the selectable marker includes fluorescence marker such as GFP and Dsred and enzymes such as LacZ.
• knock-out constructs have one or more of the sequences shown in SEQ ID NOs: 1 and 16.
• the second PrP K. O. construct (carrying zeocin instead of neomycin as the selectable marker) was then used to knock out the remaining PrP allele in those clones where one allele had been targeted with neomycin and the second PrP allele was still intact.
• zeocin R clones After PCR screening and genomic Southern analysis of isolated, zeocin R clones, the complete ablation of the PrP gene was confirmed by RT-PCR analysis to demonstrate the lack of PrP mRNA and by Western blot analysis to show complete absence of the PrP protein.
• Cells resulting from such a stable transfection performed with prion-free 293F cells routinely growing in serum-free medium and f. e. with a pcDNA3.1 construct carrying the gene of interest are seeded in semi-solid, methyl-cellulose- based medium containing an appropriate antibiotic for clone selection and a labelled antibody for detection of the highest producer clones via fluorescence. Large numbers (for example ten-thousands) of clones are then analyzed using ClonePixFL (Genetix) with respect to cell number and secreted recombinant protein in order to subsequently pick only a few hundred best producer clones.
• ClonePixFL Genetix
• the best clones are selected with respect to robustness, high growth rate, viability, scalability and production of f. e. active recombinant protein as measured by ELISA test. During this selection the number is reduced again to only a few best producing clones. Additional to the productiv- ity, correct cDNA sequence, mRNA content and cell behaviour upon fermentation are the criteria to identify the best clone(s) for subcloning. For this, cells of the selected clone(s) are re-plated, analyzed and picked with ClonePixFL, and then expanded and selected as described above.
• LB medium Tryptone 10 g, Yeast extract 5g, NaCI 10 g, dissolved into 1 I H 2 O, then autoclaved.
• FISH analysis Determination of number of chromosome 20 in 293F cells and pf293F cells:
• Cell pellet can be lysed directly or frozen for storage at -80 0 C 8. 1000 ⁇ l cold lysis buffer is added to the cell pellet
• the protein concentration of the cell lysates was measured by Bradford Assay
• Example 1 Detailed description for the first knock-out round with the PrP K. O. construct pBS_Neo_P ⁇ _R+L_Arm_2B (SEQ ID NO: 1) containing neomycin.
• PrP expression will be completely obliterated in human HEK 293 or HEK 293F cells by means of a promoter trap. This strategy (outlined in Figure 1) was designed to specifically select those cells in which the ORF of the PrP gene is replaced by the neomycin phosphotransferase ORF (except the translation initiation codon, which belongs to the PrP gene).
• Plasmid construct 2B (depicted in Figure 2) consists of 4 main components: 1.
• the vector backbone (not depicted in the figure) : pBluescript, 2.
• a "neo"( neomycin phosphotransferase) truncated cassette, consisting of the complete ORF for this gene except its own translation initiation codon followed by a transcription termination signal (see SEQ ID NO. 1).
• the neo ORF was designed to be translated from the initiation ATG codon which belongs to the PrP gene. 3.
• a "left arm” region upstream of neo with sequence identical to the PrP intron located between E2 and E3 (see SEQ ID NO. 2). 4.
• a "right arm” region downstream of neo with sequence identical to the PrP region downstream of the PrP ORF (see SEQ ID NO. 3).
• FIG. 3 Generation of the Targeting PrP K.O. Construct (pBS Neo P " R+L Arm 2B) : The generation of the targeting construct consists of three consecutive cloning steps ( Figure 3), each of which includes: a PCR amplification step to produce an "insert", a ligation step of such an insert into a vector, and transformation of the ligation mixture into E. coli and selection of correct clones by restriction enzyme digest.
• GGCAAGAATTCGC ⁇ GAGCAGTCATTATGATTGAACAAGATGGATTGCAC- GCAG -3' SEQ ID NO. 4
• 2B-Neo-R 5'- GGACCGCTCGAG-ATGCTTCCGGCTCGTATGTTGT-3' (SEQ ID NO. 5),
• the amplified product (1244 bp) was digested with EcoRI and Xhol and ligated into EcoRI/XhoI- digested pBluescript II KS+ (Stratagene). The ligation mixture was then trans- formed into One shot ToplOF' competent cells (Invitrogen). Screening was performed by EcoRI/XhoI restriction digest of plasmid DNA prepared from individual colonies. 2.
• the left arm of the targeting construct was amplified by PCR using as a template the BAC DNA clone 186 (BACPAC Resources Center, BPCR, http://bacpac.chori.org/) and the following synthetic oligonucleotides:
• 2B-L-F 5'- GGCAAGCGGCCGC-CTCTGTCTAGGAACACTGCTGTG-3' (SEQ ID NO. 6)
• 2B-L-R 5'- GGCAAGAATTC-AAAATGAAGAGGAGAACGTCAGAGTC-B' (SEQ ID NO. 7),
• 2B-R-F 5'- GGACCGCTCGAG-TGTGTACCGAGAACTGGGGTGATG-3' (SEQ ID NO. 8)
• 2B-R-R 5'- GGCGGGGTACC-GCAGAATCTCTGAGCTCACCTCAG-3' (SEQ ID NO. 9),
• the amplified product (4.6 Kb) was digested with Xhol and Kpnl and ligated into Xhol / Kpnl-digested pBS_Neo_P-_L_Arm_2B.
• the resulting ligation mixture was then transformed into SURE 2 supercompetent cells (Stratagene). Screening was performed by Xhol/Kpnl restriction digest of plasmid DNA pre- pared from individual colonies.
• pBS_Neo_P- _R+L_Arm_2B selection cassette without any promoter sequence and without its own translation initiation codon
• PrP region which in the human genome follows the PrP ORF.
• the targeting construct pBS_Neo_P-_R+L_Arm_2B was introduced into the host cells (293F, Invitrogen) using the Lipofectamin 2000CD reagent (Invitro- gen, see transfection method, 6.2). 48 h following transfection, cells were plated onto 10 cm dishes at a density between 1.25 and 1.5 x 10 6 cells. Antibiotic selection was started at the time of seeding out at concentrations rang- ing between 30 and 120 ⁇ g G418/ml. Medium exchange was performed every second/third day for 14-21 days.
• genomic DNA was prepared, either from 1 x 10 6 - I x 10 7 G418 R cells using the QIA DNAeasy tissue kit (see Molecular Biology Methods, 5.3), or by using a previously described method developed to prepare genomic DNA from ES cells plated on 96 well plates (Ramirez-Solis et al.,1992). From each genomic DNA preparation, a PCR mix was prepared containing 80-300 ng genomic DNA, Ix PCR buffer, 200 nM each oligonucleotide primer, 5 mM MgCI 2 , 200 nM dNTP and 1,33 units Expand Fidelity Polymerase (Roche). The sequence of the synthetic oligonucleotides used for screening was as follows: K.O.-Fl: 5' CGACTCAGTGTCATTCCCTGCAGTCTC 3' (SEQ ID NO.10)
• K.O.-Rl 5' CATAGCCGAATAGCCTCTCCACCCAAG 3' (SEQ ID NO.11)
• the cycle parameters were as follows: 94°C 2 min; [94°C, 15 s; 71,6°C, 30 s; 72°C 100 s] xl6 cycles; [94°C, 15 s; 71,6°C, 30 s; 72°C 100 s + 3 s longer in each successive cycle]x26 cycles; 72°C 7 min.
• Genomic DNA samples yielding a 2.3 kb PCR product indicate the presence of one or several targeted alleles in the cell population. Targeted clones were further analyzed with Southern- blot analysis.
• Genomic PCR screening ( Figure 4) : Genomic DNA was isolated from mixed cell populations or isolated cell colonies as described in D above. All PrP K. O. screening PCR reactions were performed with positive and negative controls. The template for the positive control was 200 ng genomic DNA from a cell population mix in which a targeted PrP allele had been previously detected with the PrP K. O. PCR screening method; the negative control used water instead of genomic DNA as a template. The appearance of a 2.2 kb PCR product on agarose gel after electrophoresis indicates that the corresponding cell colony bears at least one targeted PrP cell allele. With this method the clones picked by ClonePixFLwere analyzed and the results are shown in Figure 6.
• Genomic Southern analysis ( Figure 7): PrP-targeted 293F cell clones identified by genomic PCR as described above were further genetically characterized by Southern blot analysis. Following electrophoretic separation of EcoRI- digested genomic DNA and capillary transfer to Hybond+ membranes, the blots were radioactively hybridized with specifically designed DNA probes (de- picted as red arrows in Figure 5) in order to verify correct and intact integration of the neo ORF. 5'- and 3'- probes are homologous to the 5'- and 3'- external regions of the expected integration site. Neo-probe is used to verify whether the K. O. construct is integrated in the target locus or just randomly.
• Neo probes were generated by PCR amplification using the BAC DNA clone 186 described in section B.2 of this Example.
• the Neo probe was amplified from plasmid pcDNA3.1( + ) which includes a neomycin cassette, summarized in the following table:
• the genomic Southern strategy with construct pBS_Neo-_P-_L+R_Arm_2B is depicted in Figure 5.
• targeted PrP clones show a 4.2 kb band with the 5'-DNA probe and a 6.5 kb band with the 3'-DNA probe, while the wild-type band from the non-targeted allele , detected with either probe, is 10.8 kb.
• the Neo-probe a 6,5kb DNA band should be detected for PrP targeted allele and no band should be detected for wild-type 293F cells.
• FISH analysis ( Figure 8) Human PrP gene is located on chromosome 20. 293F cells were hybridized with a WCP (whole chromosome painting) probe for painting of chromosome 20. This FISH analysis shows that 25% of the 293F wild type cells have 2 copies of chromosome 20, while 75% of 293F cells bear 3 copies of chromosome 20. Technically it is very difficult and almost not possible to knock out 3 PrP genes using the current technologies. Therefore, FISH analysis with chr20 WCP painting probe helps to distinguish if the cell clones already bearing one PrP targeted allele have two or three chromosome 20. Additionally, a BACDNA clone 186 probe, which spans over 100kb, including the PrP gene, and is a part of chromosome 20, was also used for FISH hybridization for detection of possible chromosome deletions and translocations.
• K.O. 1 and K.O. 6 were further analyzed by ELISA to determine if their PrP protein levels are about half of wild type 293F cells.
• Example 2 Detailed description for the second knockout round with a PrP K.O. construct containing zeocin.
• the pBS_Neo_P-_R+L_Arm_2B plasmid was digested with EcoRI and Xhol and ligated with PCR-amplified Zeocin cassette, which contains on its ends restric- tion sites of EcoRI and Xhol.
• the plasmid generated from this ligation was called pBS_Zeo_P-_R+L_Arm (Fig. 10) (SEQ ID NO. 16) and used for targeting the second PrP allele.
• This second PrP K. O. construct pBS_Zeo_P- _R+L_Arm was linearized with BamHI for transfection as described in B.4.
• the second PrP K.O. construct, pBS Zeo P R+L Arm into PrP K.O. cells with one allele of PrP gene The second PrP K.O. construct, pBS_Zeo_P_R+L-Arm was introduced into identified targeted cells with one targeted PrP allele using Lipofectamin 2000 CD reagent (see transfection method page 19). 48h after transfection, cells were plated onto 10 cm dishes or further cultured in suspension in 125 ml shaker flasks at a density between 1.25 and 1.5xlO 6 cells/ml.
• G418 and zeocin were added to culture at the following concentrations: G418 0-30 ⁇ g/ml and zeocin of 0-30 ⁇ g/ml. Medium exchange was performed every second/third day for 14 - 30 days.
• the PCR screening strategy was similar to that of Example ID except that the reverse primer for PCR was designed to be homologous to the zeocin ORF.
• a PCR mix was prepared containing 20-300 ng genomic DNA, Ix PCR buffer, 200 nM each oligonucleotide primer, 1,25 mM MgCI2, 200 nM dNTP and 1,33 units Expand Fidelity Polymerase (Roche).
• the sequence of the synthetic oligonucleotides used for screening was as follows:
• Zeo-K.O.-R2 5' CGAAGTCGTCCTCCACGAAGTC 3' (SEQ ID NO. 20)
• the cycle parameters were as follows: 94°C 4 min; [94°C, 15 s; 63°C, 30 s; 72°C 100 s] xl6 cycles; [94°C, 15 s; 63°C, 30 s; 72°C 100 s + 3 s longer in each successive cycle] x24 cycles; 72 0 C 7 min.
• Genomic DNA samples yield- ing a 2.3 kb PCR product indicate the presence of one or several targeted alleles in the cell population.
• Genomic PCR screening ( Figure 11) : To identify if the cells or cell mix population bear two targeted PrP alleles, two independent PCR screening should be performed. PCR primer pair Zeo-K.O.-F2 and Zeo-K.O.-R2 was used to prove targeted integration of the second PrP Ko. O. construct (pBS_Zeo_P- _R+L_Arm), while primer pair K. O. -Fl and K.0.-F2 proves targeted integration of the first PrP K. O. construct (pBS_Neo_P-_R+L_Arm_2B).
• Genomic DNA was isolated from mixed cell populations or isolated cell colonies as described in C above. All PrP K. O. screening PCR reactions were performed with positive and negative controls. The template for the positive control was
• the genomic Southern strategy with construct pBS_Zeo-_P-_L+R_Arm is depicted in Figure 11.
• targeted PrP clones showed a 4,2 kb band with the 5'-DNA probe and a 5,7 kb band with the 3'-DNA probe, while the wild-type band from non-targeted alleles, detected with either probe, is 10.8 kb.
• Zeo-probe a 5,7 kb DNA band should be detected for PrP targeted allele and no band can be detected for wild-type 293F cells.
• Example 3 Serum-free transfection of the prion-free 293F cell line pf293F for generation of prion-free recombinant proteins.
• PrP KO clones with one PrP targeted allele were transfected with PCDNA3.1-FVIII (SEQ ID NO. 26), pcDNA3.1-FIX (SEQ ID NO. 17) and pcDNA3.1-hyg( + )-G-CSFb (SEQ ID NO. 25) vectors to express human FVIII, FIX and G-CSFb proteins.
• pf293F cells transfected as described above were seeded in semi-solid methyl- cellulose based medium containing an appropriate antibiotic, for selection of clones, and a labelled antibody for detection of the highest producer clones via fluorescence.
• Large numbers (thousands) of clones were analyzed using ClonePixFL (Genetix) with respect to the cell number and to secretion of the target FIX protein in order to subsequently pick only a few hundred best FIX producer clones.
• ClonePixFL allows picking of fast growing clones, which are high producers only, originated from single cells. The picked cells are expanded in microtiter plates and later in spin tubes, cell culture flasks and fermenters under serum-free conditions for the complete procedure.
• FIX production clones identified by the method described above were cultured in serum-free Freestyle 293 expression medium.
• the target proteins were isolated and purified according to standard procedures. Additionally, for pro- duction of safe therapeutics, the optimised purification procedure as disclosed in PCT/EP 2006/061148 including f. e. SD-treatment could be utilized.
• Pf293F cells will also be used for serum-free production of recombinant human FVIII and G-CSF with expression vector pcDNA3.1-hyg( + )-G-CSFb (SEQ ID No. 25) and pcDNA3.1-FVIII (SEQ ID No. 26).
• SEQ ID NO.25 pcDNA3.1-hyg(+)-G-CSFb
• SEQ ID NO. 26 pcDNA3.1-FVIII Molecule: 9975 bps DNA Circular

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