Patent Grant
7038107
Not applicable.
1. Field of the Invention
This invention relates to compositions and methods for detecting the presence of endogenous retroviruses, in particular, the detection of porcine endogenous retroviruses (PERV) in tissues useful as a xenograft.
2. General Background and State of the Art
The most common source of tissue used today as donor tissue for transplantation is the allograft (same species, different person). However, there are insufficient resources of human organs and cells for use as donor tissue. The shortage of human donor material has resulted in alternative solutions for transplantation. Xenotranplantation, which is the use of living tissue from non-human animals is one viable alternative. However, when using alternative sources, ethical, practical, biological and economic concerns must be considered. Therefore, among the animal species most suitable for use as a xenotranplant is the pig.
Examples of porcine tissue already being tested in clinical trials include fetal pig pancreatic islet cells for treating diabetes (Groth et al., Lancet 1994), pig neuronal cells for treating Parkinson's disease (Deacon et al., Nat Med 1997) and extracorporeal (ex vivo) pig liver or kidney perfusion for treating liver (Foley et al., Transplantation 2000, Levy et al., Transpl. 2000. 69: 272) or kidney failure (Breimer et al., Xenotransplantation 1996). However, as the potential for success increases the use of porcine tissue as a resource, the potential for introducing an infectious agent from the pig into the recipient becomes an increasing concern. Although risks associated with some pathogens can be reduced by breeding for and using specific-pathogen-free (SPF) animal colonies, this approach is not feasible for preventing infection from endogenous retroviruses, because these pathogens exist in the germine of all pigs.
Porcine Endogenous Retrovirus (PERV) is a C-type retrovirus that is permanently integrated in the pig genome. PERVs exist in the pig genome at an estimated 25–50 copies per cell. Early scientific reports dating back to the 1970's indicated that some porcine (pig) cells grown in culture produced type C retrovirus particles. This suggested their potential to be infectious. However, other studies of cells cultured directly from pig tissue (primary culture) showed no evidence of infectious potential. More recent reports indicate that there is a low frequency of PERV infection of human cells that are co-cultured with a pig cell line, PK15 (Le Tissier et al., Nature 1997; Patience et al., Nature Medicine, 1997). This reemphasized the concern regarding potential risk of PERV infection to human recipients of porcine tissue xenografts. Augmenting this risk is the use of immunosuppressive therapies necessary for preventing graft rejection by a recipient. Suppression of the recipient's immune system may also significantly increase the recipient's susceptibility to infection by PERV. Therefore, there is a need for methods to detect and monitor porcine tissues and cells for the presence of infectious PERV.
To date, there are three classes of PERV (PERV-A, PERV-B, and PERV-C). The differences between the three classes are primarily based on sequence differences in their envelope gene region. Recent identification of PERV sequences allows for development of molecular based detection methods. For example, methods that can detect specific sequences of DNA such as the polymerase chain reaction (PCR) can be used to identify the presence of PERV in a tissue sample. Keeping in mind that all pig genomes have PERV sequences, but that not all PERV are infectious, prescreening of a transplant tissue merely for presence of a PERV sequence is not a sufficient indicator of its infectious potential. Therefore, current methods for detecting the presence of PERV are limited by their inability to determine which PERV loci are infectious.
Thus, there is a need for methods and compositions capable of reducing the risk of transmission of PERV from porcine tissues suitable for use as xenografts. Particularly needed are compositions and methods for detecting the presence of infectious PERV in a biological sample.
The present invention provides compositions and methods for detecting porcine endogenous retroviruses (PERV). These methods and compositions are particularly effective in detecting the presence of PERV loci capable of producing infectious virus. The present invention provides methods for detecting the presence of PERV in samples useful for xenotransplantation.
In one embodiment, the invention provides compositions that are capable of detecting potentially infectious PERV using nucleic acid sequences comprising porcine sequences flanking infectious PERV insertion sites. The unique 3′ flanking sequences or unique 5′ flanking sequences can be used to provide nucleic acid probes that are specific for potentially infectious PERV loci. In addition, nucleic acids sequences and subsequences thereof from the PERV genome of three infectious PERV loci, G3, G19, and G28 can be used to provide nucleic acid probes, including primers (SEQ ID NO:137–139). Such genomic sequences can also be used to provide nucleic acid probes that are specific for potentially infectious PERV loci.
Accordingly, a unique 3′ flanking sequence from a PERV locus can include any one of SEQ ID NO:6–35 or any nucleic acid sequence capable of hybridizing under suitably stringent conditions to any one of SEQ ID NO:6–35. Furthermore, the invention also provides nucleic acid sequences having suitable sequence identity, such as at least 80% sequence identity, to a porcine 3′ end flanking sequence of an infectious PERV insertion site. The provided nucleic acid sequences and probes can be used for diagnosis, monitoring or screening of specific PERV loci in cells, tissues, or organs suitable for use as a xenotransplant.
The provided porcine sequences flanking potentially infectious PERV integration sites can be used as probes in methods for detecting presence of potentially infectious PERV in a biological sample. Suitable detection assays include use of the PERV probes in Southern Blot Analysis, PCR Analysis, or other molecular biological assay comprising formation of a PERV genomic target region:probe duplex and detection of the target region:probe duplex in the sample.
The invention further provides methods for making probes suitable for detecting the presence of potentially infectious PERV. Such methods include isolation of nucleic acid sequences which flank a PERV integration site and identification of the sequences as unique flanking sequences. Isolation of nucleic acid sequences flanking a PERV integration site can be by use of a conserved PERV sequence; or use of a conserved PERV sequence derived from the envelope region of a PERV sequence. The unique flanking sequences can be a unique 3′ flanking sequence or, alternatively, can be a unique 5′ flanking sequence. The unique 3′ flanking sequences or unique 5′ flanking sequences can be identified using DNA sequence analysis. Therefore, the invention also provides methods for detecting sequences capable of hybridizing to a unique 3′ flanking sequence or a unique 5′ flanking sequence.
Such methods include isolation of nucleic acid sequences which flank a PERV integration site and identification of the sequences as unique flanking sequences. Such methods include isolation of nucleic acid sequences which flank a PERV integration site and identification of the sequences as unique flanking sequences. The unique flanking sequences can be a unique 3′ flanking sequence or, alternatively, can be a unique 5′ flanking sequence suitable for detecting presence of potentially infectious PERV.
Therefore, the compositions of the present invention also provide methods for reducing the risk of transmission of PERV from a xenotransplant tissue to a host or recipient.
Accordingly in another aspect of the invention, methods for selecting animals free of specific PERV loci are provided. Pigs having a negative profile for a specific PERV locus or a potentially infectious PERV can be used to breed a pig for use as a xenograft donor. A selective breeding method would include determination of a pig's PERV allele polymorphism profile. Selection of those pigs having a profile that is negative for a specific PERV locus can then be made. Those having a negative PERV locus profile can be bred to obtain offspring whose genome are free of one or more specific PERV loci. An animal produced by the present invention can therefore be used as a source of xenograft tissue that is free of potentially infectious PERV loci, such as a G3 (SEQ ID NO:8), a G19 (SEQ ID NO:24) or a G28 (SEQ ID NO:33) locus.
The present invention includes compositions and methods for detecting porcine endogenous retrovirus (PERV) and methods for making probes suitable for use in detecting PERV. In particular, compositions comprising molecular probes such as nucleic acid sequences specific for infectious endogenous retroviruses are provided. Also provided are methods of making and using such probes as well as assays employing such probes. The present invention is applicable to the breeding or selection of donor transplant tissue, for example, porcine tissue, that is free of infectious endogenous retroviruses. The foregoing strategy can be utilized with any infectious or potentially infectious endogenous retroviral sequence to practice the method of the invention and accordingly, the present invention is not specifically limited to compositions and methods of making and detecting PERV loci alone nor only those PERV loci specifically disclosed herein. While the present invention is not so limited, an appreciation of various aspects of the invention will be gained through a discussion of the examples provided below.
PERV belongs to the mammalian type C class of retroviruses. Attachment of the retrovirus to a host cell prior to infection is mediated by viral surface proteins, which are encoded by the envelope (Env) gene region of the retroviral sequence. These viral surface proteins bind host cell surface proteins. The Env gene proteins play a role in determining host range specificity. There are three classes of PERV identified thus far based on their Env gene sequences. These sequences have been published and deposited (Perv-A=Genbank Accession No: AF038601; Perv-B=EMBL Accession No: PERY17013; PERV-C=Genbank Accession No: AF038600).
PERV exists in the genome of all pigs. As discussed previously, the ability of some cultured pig cells to infect human cells in vitro raises the concern that pig tissue used for xenotransplantation may be capable of transmitting infectious PERV to the recipient. Although co-culture assays have identified some pig cells that are infectious by their ability to infect human cell lines in culture, this technique cannot be used as a reliable screening assay due to its low sensitivity. For example, co-culture with activated lymphocytes or hepatocytes from one source of transgenic pig failed to show any evidence of productive infection. This could indicate either that the particular line of transgenic pig did not have infectious PERV, that the sequences were not activated, or that PERV sequences from these pigs are not able to infect the particular line of human cells used.
Multiple PERV proviral sequences exist within a pig genome. The degree of homology within these sequences is high, making them difficult to distinguish by sequence polymorphism. One embodiment of the present invention is based upon each PERV proviral integration into the pig genome representing a unique event. PERV clones were isolated from a genomic library constructed using DNA from a transgenic pig in our herd, and unique sequences flanking the 3′ and 5′ region of each PERV gene were identified as described more fully in the examples below. The pigs are known and described as 603-57 transgenic line (Byrne et al. 1997 Transplantation 63:149–155). The pigs carry the human CD59 and human CD55 transgenes. Pig d711, a DNA donor for a porcine genomic library was obtained by crossing a transgenic founder pig (603-57) to a nontransgenic sow. Both pigs were from a Camborough 15 Line (Pig Improvement Company, Franklin, Ky.). A Camborough 15 pig is 25% Large White, 25% Landrace, and 50% Duroc. Alternatively, PERV clones can, if desired, be isolated from a genomic library using DNA from any pig or any transgenic pig whose organs or tissues are suitable for use in xenotransplantation.
As used herein, the term “unique”, “unique flanking” or “unique flanking sequence” refers to those sequences from a pig genome which flank a PERV integration site and which can be distinguishable from non-PERV flanking genomic sequence based on flanking sequence polymorphism.
Since those PERV loci that have an envelope gene are competent to be infectious, this region of the PERV sequence was used to construct probes for isolating each PERV sequence. Based on known PERV envelope sequences, conserved envelope sequence probes (env-cons) which correspond to the transmembrane domain of the PERV envelope gene were constructed: PERV-A (1881–2133 bp, Y12238) (SEQ ID NO:1); PERV-B (2572–2824 bp, Y12239) (SEQ ID NO:2); and PERV-C (7227–7479 bp, AF038600) (SEQ ID NO:3). Additional probes used to screen the genomic library included: PERV-A (94–2133 bp, Y12238) (SEQ ID NO:4) and PERV-B (794–2823, Y12239) (SEQ ID NO:5). However, any region of a PERV sequence, such as for example, a sequence from a gag, pol, signal or LTR region of a PERV sequence could be used to construct probes for isolating a PERV locus.
All probes were initially generated by PCR amplification as described in the examples below. Any method, however, for nucleotide sequence replication can be used to generate a PERV probe. Such methods include, for example, ligase-chain reaction, isothermal amplification, or use of more basic cloning techniques such as use of cloning vectors or plasmids, and propagation from a bacterial stock. For additional details and explanation of nucleotide sequence amplification or replication techniques, see Ausubel et al. Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995); US Dept Commerce/NOAA/NMFS/NWFSC Molecular Biology Protocols (URL:http://research.nwfsc.noaa.gov/protocols.html); or Protocols Online (URL: www.protocol-online.net/molbio/index.htm).
PERV envelope probes were used to screen a pig genomic library and isolate PERV loci. Sequence analysis of each clone into the flanking genomic DNA identified each clone as unique or not unique. As set forth in Examples 2–4, the PERV clones (G3–25 (SEQ ID NO:8); G19-A45 (SEQ ID NO:24)and G28-402A (SEQ ID NO:33)) were made. Therefore, the invention provides methods of obtaining clones and their use in constructing probes for identification of unique PERV flanking sequences.
The invention further provides newly identified and isolated nucleotide sequences (SEQ ID NOS:6–35; and 101–118) flanking integrated PERV genes. Probes corresponding to the 3′ flanking PERV integration sequences were then obtained by amplification using 5′ primers (5′G1–5′G30) (SEQ ID NOS:36; 38; 40; 42; 44; 46; 48; 50; 52; 54; 56; 58; 60; 62; 64; 66; 68; 70; 72; 74; 76; 78; 80; 82; 84; 86; 88; 90; 92; and 94) and 3′ primers (3′G1–3′G30) (SEQ ID NOS:37; 39; 41; 43; 45; 47; 49; 51; 53; 55; 57; 59; 61; 63; 65; 67; 69; 71; 73; 75; 77; 79; 81; 83; 85; 87; 89; 91; 93; and 95). Primers used as pairs for amplification are listed in Example 2, Table 1.
The size or length of genomic flanking sequence sufficient to allow for identification of a unique PERV locus was approximately 300–500 bp of DNA downstream (for identification of unique 3′ flanking sequence) or upstream (for identification of unique 5′ flanking sequence) of each individual PERV gene. However, as would be known to one skilled in the art, a length of approximately less than 100 bp, or greater than 1000 bp, or of 100–2000 bp, can also be used if necessary, depending upon the extent of polymorphism in the flanking region, the degree of comparison desired, or the level of sequence distinction required.
A flanking sequence was identified as unique by DNA sequence analysis and comparison. Sequence reactions and analysis was performed using standard sequence reaction kits (PE Applied Biosystems, Foster City, Calif.), but can be performed by other methods well known in the art and described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 1989) or in Innis et al., PCR Strategies (Academic Press, Inc.: NY., 1995). In some cases, sequence information was obtained from the genomic DNA flanking the upstream (5′ region) of the PERV gene. As would be apparent to one skilled in the art, if sequence analysis is performed on 5′ flanking sequence, then sequences would be compared to other 5′ flanking sequence and not 3′ flanking sequences. The size or length of flanking sequence sufficient for DNA sequence analysis and comparison was similar to that for the 3′ flanking regions.
The unique 3′ flanking sequences from each PERV locus provides locus specific flanking probes. Based on the unique 3′ flanking sequence from each PERV locus, a locus specific probe was obtained for each locus (SEQ ID NOS:6–35) also referred to herein as G1–G30, and as described more fully in the Examples below. As used herein, a “unique 3′ flanking sequence” is used to refer to a genomic pig sequence that is a unique sequence flanking the 3′ end of a PERV integration locus. A unique 3′ flanking sequence as used herein also refers to any nucleic acid sequence having at least about 80%, 90%, 95% or greater DNA sequence identity or homology with the 3′ flanking sequence shown in Table 1, which sets forth the 3′ probe and primer sets for each PERV locus (G1–G30); (SEQ ID NOS:6–35).
As used herein, a “unique 5′ flanking sequence” is used to refer to a genomic sequence flanking the 5′ end of a PERV integration locus. A unique 5′ flanking sequence as used herein also refers to any nucleic acid sequence having at least about 80%, 90%, 95% or greater DNA sequence identity or homology with a 5′ flanking sequence (SEQ ID NOS:101–118) corresponding to PERV loci, or as identified using the methods described herein.
The invention further provides those endogenous retroviral sequence loci having infectious potential. The ability of each individual PERV gene to produce infectious virus was assessed. Plasmid containing one PERV gene as well as a selectable drug resistance marker was transfected into 293 cells. The cell supernatant from drug-resistant clones was assayed for RT activity. Any clone which gave rise to measurable RT activity was then analyzed for the ability to produce infectious retrovirus by co-culturing the supernatant with fresh 293 cells. Cell supernatant from this culture was assayed for RT activity, and those cells collected on a weekly basis were analyzed to detect PERV integration. Thus far, three PERV clones, G3–25 (SEQ ID NO:8); G19-A45 (SEQ ID NO:24), and G28-402 A (SEQ ID NO:33) when transfected into 293 cells, result in RT activity in the supernatant. Additional endogenous retroviral sequences with infectious potential from other animals can also be identified using the probes, screening methods and strategy described above to identify novel endogenous retroviral integration sites.
The invention further provides assays for detecting the presence of specific endogenous retrovirus that are potentially infectious in a sample, including organs, tissues, cells, and fluids. Samples identified as being positive or negative for specific infectious endogenous retroviral sequence are determined by testing for presence or absence of novel flanking sequences, such as for example, a unique 3′ flanking or a unique 5′ flanking sequence associated with a specific PERV gene.
Analysis of PERV loci or any other endogenous retroviral sequence locus can be by methods such as Southern blot analysis, conventional PCR amplification. See, e.g., Innis et al., PCR Strategies (Academic Press, Inc.: NY., 1995); Dieffenbach et al., PCR Primer: A Laboratory Manual (New York: Cold Spring Harbor Press, 1995), denaturing gradient gel-electrophoresis (Myers, et al., 1987. Meth. Enzymol. 155: 501), single-strand conformational analysis (Hayashi, 1992. Genet Anal Biomol E 9: 73), ligase-chain reaction (Barany. 1991. Proc Natl Acad Sci 88: 189), isothermal amplification (Fahy et al. 1991. PCR Methods Appl 1: 25), branched chain analysis (Urdea. 1993. Clin Chem 39: 725), and signal amplification techniques such as Third Wave's linear amplification. DNA sequence analysis may also be achieved by detecting alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Samples containing PERV insertions adjacent to a unique 3′ flanking sequence or a unique 5′ flanking sequence can also be visualized by high resolution gel electrophoresis or distinguished according to differences in DNA sequence melting points. See, e.g., Myers et al., Science. 230: 1242 (1982). Methods for detecting presence of specific sequences include detection techniques such as fluorescence-based detection methods, immune-based assays such as RIA, antibody staining such as Western blot analysis or in situ hybridization, using appropriately labeled probe, based on the sequences provided herein. Using the methods and strategy described hereinabove, any unique 3′ flanking sequence or unique 5′ flanking sequences in addition to those specifically disclosed herein can be identified and used as probes for detection or analysis of PERV presence.
PERV Allele Polymorphism
The invention also provides a method to screen for the existence of PERV with potential to infect in a sample by determining PERV loci allele polymorphism. Identification of allelic polymorphism in a sample allows for selection of animals suitable for use either directly as a xenograft tissue donor or as a breeder to provide a source of xenograft tissue that is negative for, or free of PERV sequence that are infectious or have potential to be infectious. One method for detecting presence of a PERV locus is analysis of allele polymorphism by Southern blot using a unique flanking sequence as a probe, such as a unique 3′ flanking sequence or a unique 5′ flanking sequence. Using Southern blot analysis, a different banding pattern will emerge if PERV is present depending upon whether PERV is present on both alleles, on one allele, or on neither allele. Southern analysis of three infectious PERV loci, G3 (SEQ ID NO:8, see also SEQ ID NO:137 (complete G3 PERV genome),
Sequences useful for constructing probes suitable for use in detecting presence of PERV include any one of G1–G30 (SEQ ID NOS:6–35) or any nucleic acid sequence having at least about 80%, 90%,95% or greater sequence identity or homology with a unique 3′ flanking sequence (SEQ ID NOS:6–35) as determined by a Blast search. “Percent (%) sequence identity” or “percent (%) sequence homology” with respect to sequences identified herein is defined as the percentage of nucleic acid residues in a candidate sequence that are identical with the nucleic acid residues disclosed herein (any one of SEQ ID NOS:6–35), after aligning the sequences and introducing gaps, if necessary to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Methods for performing sequence alignment and determining sequence identity are well known in the art, may be performed without undue experimentation, and calculations of % identity values may be obtained for example, using available computer programs such as WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460–480 (1996). One may optionally perform the alignment using set default parameters in the computer software program (Blast search, MacVector and Vector NTI).
Based upon the restriction map of a particular locus, a banding pattern can be predicted when the Southern blot is hybridized with a probe which recognizes a unique flanking sequence, such as a unique 3′ flanking sequence. The level of stringency of hybridization used can vary depending upon the level of sensitivity desired, a particular probe characteristic, such as probe length and/or annealing temperature, or degree of homology between probe sequence and genomic sequence flanking a 3′ or a 5′ region of a PERV locus. Therefore, considerations of sensitivity and specificity will determine stringency of hybridization required for a particular assay.
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperatures. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al. Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995) or Protocols Online URL: (www.protocol-online.net/molbio/index.htm).
“Stringent conditions” or “high-stringency”, as defined herein, may be identified by those that use low ionic strength and high temperature for washing, for example 0.1×SSC, 0.2% SDS @65–70° C. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
“Moderately-stringent conditions” may be identified as described by Sambrook et al., supra, and include the use of washing solution and hybridization conditions (e.g. temperature, ionic strength, and % SDS) less stringent that those described above. Onbe example of moderately stringent conditions is 0.2×SSC, 0.1% SDS @58–65° C. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. The skilled artisan will recognize how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length, degree of homology between probe and target site and the like. Therefore, in addition to the unique 3′ flanking sequences and unique 5′ flanking sequences described herein, it is contemplated that additional or alternative probe sequences which vary from those specifically disclosed herein (SEQ ID NOS:6–35) will also be useful in screening for PERV loci having infectious potential.
Another method to determine the existence of an allele containing or missing a specific PERV integration utilizes PCR analysis. For example, presence or absence of a PERV locus in a sample can be determined by detecting a change in size of the amplified product in comparison to the expected size product from a known genotype. Genomic DNA from a test sample is analyzed with specific primers that flank a particular PERV locus. Suitable primers include SEQ ID NOS:36–95; useful as primer pairs as disclosed in Table 2.
The present invention includes methods of detecting the potential for infection by a specific PERV by detecting presence of a PERV locus-specific integration site using novel flanking sequences. The sample to be tested or analyzed may be obtained from any biological source known to or suspected to carry potentially infectious PERV, and is preferably taken from an animal prior to use as a donor. For example, the sample may be a cell sample, tissue sample, or biological fluid, such as blood, urine, semen, saliva, sputum, tissue culture fluid, ascitites fluid, synovial fluid, and the like. The sample may also be a hair sample where DNA from the follicle can be isolated for analysis. A laboratory research sample such as a cell culture or embryo culture can also be used as the test sample. The sample is collected and processed for genomic DNA using methods well known to those skilled in the art.
The molecular based assays described herein, such as the PCR based assay, are ideal in that they have a high degree of sensitivity and specificity, thereby reducing the chance for false-positive or false-negative results. Alternatively, the unique flanking sequences (unique 3′ flanking sequences or unique 5′ flanking sequences) can be used to develop probes for use in a DNA detection method such as, for example, a conventional Southern blot assay. Probes suitable for such screening assays can comprise any one of the unique flanking sequences disclosed herein. For example, a probe suitable for use in a Southern blot assay would include a unique 3′ flanking sequences such as SEQ ID NO:8; SEQ ID NO:24 or SEQ ID NO:33. Furthermore, detection of a potentially infectious PERV locus could be performed indirectly by use of sequences capable of hybridizing or complexing to the unique flanking sequences described herein. Methods for performing Southern blot assays are well known in the art and described in Sambrook et al., supra. Therefore, the invention also provides an assay kit for the detection of PERV loci in a sample. The assay kit will preferably contain necessary reagents and tools for reacting the sample with a PERV detection probe.
The provided porcine sequences flanking potentially infectious PERV integration sites can also be used to generate pigs whose genome lacks potentially infectious PERV. Such “knock-out” pigs whose genome are free of one or more specific PERV loci, can be produced using the unique 3′ flanking sequences or unique 5′ flanking sequences with standard recombination-based techniques well-known in the art and described, for example in te Riele et al., Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. U.S.A. 89:5128 (1992) and Rulicke T. Transgenic technology: an introduction, Int J. Exp Pathol 77:243 (1996). The “knock-out” pigs having a negative profile for a specific PERV locus or a potentially infectious PERV can be used directly as a source of donor tissue or can be used to further breed pigs for use as a xenograft donor.
The present invention is further illustrated by the following examples.
Commercially available reagents referred to in the Examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, is human 293 cells (ATCC: CRL-1573). Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described in: Sambrook et al., supra; Ausubel et al. Current Protocols in Molecular Biology On CD-ROM (Green Publishing Associates and Wiley Interscience, N.Y., 1993); Ausubel et al. Short Protocols in Molecular Biology. 4th Edition (Wiley Interscience, N.Y., 1999); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture. 1987; Cell and Molecular Biology Online (URL: www.cellbio.com/protocols.html); Protocols Online (URL: www.protocol-online.net/molbio/index.htm).
The following is provided for exemplary purposes only and to further aid in the understanding of the invention. As is apparent from the disclosure provided herein, one skilled in the art can make, use or obtain any clone or clones to unique flanking sequences of PERV loci using the methods as exemplified below and described herein.
Cloning of Unique Flanking Sequences
Construction of Pig Genomic Library
All unique 3′ flanking sequence and unique 5′ flanking sequence clones were isolated from a genomic pig library constructed as follows:
High molecular weight (>100 kb) genomic liver DNA from a transgenic (CD59/DAF) male pig (for example, an F2 outcross consisting of Large White, Landrace, and Duroc) was partially digested with Sau3Al, and 9–24 KB fragments were isolated by sucrose gradient ultracentrifugation. The fragments were cloned into a Lambda FIX II vector (Stratagene, Calif.) to construct a pig genomic library.
The amplified library titer was 5.2×109 pfu/ml. Nine million phage were screened using several different probes, all of which detect the PERV envelope gene. It is the envelope sequence of a PERV gene that is responsible for its ability to infect a cell. Envelope probes were chosen to restrict isolation of PERV sequences to only those PERV loci having infectious potential, and therefore to those sequences which carry an envelope gene.
Probes Used to Screen Porcine Genomic Library
PERV loci in the transgenic pig library that contain a PERV envelope gene were isolated. Those PERV loci that have an envelope gene will be competent to be infectious. Therefore, various probes were generated using published PERV envelope sequences. See, for example, Akiyoshi et al., (J. Virology. 72:4503 1998) and Le Tisser et al., (Nature. 389:681, 1997).
Referring to
Primers were designed using MacVector primer design program (Oxford Molecular, Madison, Wis.) and synthesized by Gibco BRL (Life Technologies, Inc., Rockville, Md.) to amplify the env-cons probe by PCR. Primers synthesized:
The PCR Reaction Used:
PCR Program:
Initial denaturation for 3 min @95° C., followed by 35 cycles of:
extension for 7 min at 72° C.
Primers were designed using MacVector primer design program (Oxford Molecular, Madison, Wis.) and synthesized by Gibco BRL (Life Technologies, Inc., Rockville, Md.) to amplify the envelope gene from either PERV-A or PERV-B using PCR:
The PCR Reaction Used:
PCR Program:
Identification of PERV Loci By Unique 3′ Porcine Genomic Flanking Sequences:
Multiple PERV proviral sequences exist, and have similar nucleotide sequence, making them difficult to distinguish by sequence polymorphism. However each PERV proviral integration into the pig genome represents a unique event. PERV loci were identified as unique based on sequence analysis of genomic DNA flanking each PERV gene. Approximately 300–500 bp of genomic DNA downstream (3′flanking) of each individual PERV gene was sequenced using an ABI automated sequencer (Perkin Elmer, Foster City, Calif.). In some cases, sequence information was obtained from the genomic DNA flanking the upstream 5′ flanking region of the PERV gene as well, which is described below.
Identification of PERV Loci By Unique 5′ Porcine Genomic Flanking Sequences
The strategy for obtaining unique 5′ flanking sequences was essentially similar as that used for obtaining unique 3′ flanking sequences, with the difference that reverse primers, such as LTR34C (SEQ ID NO:99) or LTR100C (SEQ ID NO:100), were used to sequence DNA flanking the upstream 5′ flanking region of PERV loci. Referring to
Sequence reactions were done using ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kits from PE Applied Biosystems (Perkin Elmer, Foster City, Calif.). A 20 μl sequence reaction consisted of 30–1000 ng of a DNA template (lambda DNA, plasmid DNA or PCR product), 8 μl terminator ready reaction mix, and 4 pmol of each primer. The PCR sequence programs were: 96° C. for 1 min, 35 cycles of 96° C. for 10 sec, 52° C. for 10 sec and 60° C. for 4 min. The PCR products were purified using a centri-sep column according to manufacture's protocol (Princeton Separations, Inc., Adelphia, N.J.), and sequences were analyzed by an ABI Prism 310 genetic Analyzer (Perkin Elmer, Foster City, Calif.).
3′ Genomic Flanking Probes
Based on the unique 3′ flanking sequences from each PERV locus, locus-specific 3′ flanking probes were amplified using specific primers (SEQ ID NOS:36–95). Each probe was subcloned into a TA cloning vector (Invitrogen, Carlsbad, Calif.), and sequence-confirmed.
Blast Search of 3′ Flanking Sequences Against Databases
The sequences of 3′ flanking probes of all PERV loci identified as described above, were searched against 11 databases (nr, dbests, dbsts, mouse ests, human ests, other ests, pdb, patents, epd, gss, and htgs, Altschul et al., 1997. Nucl. Acids Res. 3389–3402). The following PERV loci had no significant homologies with any of the sequences deposited in the above identified databases: G1–8, G10–12, G14–16, G19–22, G24–26 and G28–30. The PERV G6, G9, G13, G17, G18, G23, and G27 had homologies with certain database sequences as listed below in Table 3.
Cloning of a Unique 3′ Flanking Sequence, G3–25 (SEQ ID NO:8)
The following illustrates how one skilled in the art would make or obtain a PERV clone having a unique 3′ flanking sequence. Given the following description and teachings, one skilled in the art can apply the following methods to obtain any PERV clone described herein and is not intended to be limited to isolation of a specific PERV clone. A lambda library was constructed as described in Example 1. A PERV clone was isolated from a lambda FIX II vector using the env-cons probe (SEQ ID NO:1). In order to distinguish a 3′ flanking sequence as distinct and unique (i.e. as a PERV locus), the PERV lambda clone was amplified using the primer pairs env5(2) and T3 or T7 (see
The G3–25 clone, containing a unique 3′ flanking sequence suitable for identification of potentially infectious PERV, was then subcloned into a pZerO-2 vector (Invitrogen, Calif.) at the vector's Not I site (
Once subcloned, G3–25 containing plasmids were transfected into human 293 cells (ATCC: CRL-1573) and positive transfectants obtained following G418 selection. Supernatant from the 293 cells containing a G3–25 clone was then co-cultured with hygromycin resistant 293 cells (Hygr) and grown under hygromycin selection.
Cloning of a Unique 3′ Flanking Sequence, G19-A45 (SEQ ID NO:24)
The following illustrates how one skilled in the art would make or obtain a PERV clone having a unique 3′ flanking sequence. Given the following description and teachings, one skilled in the art can apply the following methods to obtain any PERV clone described herein and is not intended to be limited to isolation of a specific PERV clone. A lambda library was constructed as described in Example 1. A PERV clone was isolated from a lambda FIX II vector using the PERV-A probe (SEQ ID NO:4). In order to distinguish a 3′ flanking sequence as distinct and unique (i.e. as a PERV locus), the PERV lambda clone was amplified using the primer pairs env5(2) and T3 or T7 (see
The G19-A45 clone, containing a unique 3′ flanking sequence suitable for identification of potentially infectious PERV, was then subcloned into a pZerO-2 vector (Invitrogen, Calif.) at the vector's Not I site (
Once subcloned, G19-A45 containing plasmids were transfected into human 293 cells (ATCC: CRL-1573) and positive transfectants obtained following G418 selection. Supernatant from the 293 cells containing a G19-A45 clone was then co-cultured with hygromycin resistant 293 cells (Hygr) and grown under hygromycin selection.
Cloning of a Unique 3′ Flanking Sequence, G28-402A (SEQ ID NO:33)
The following illustrates how one skilled in the art would make or obtain a PERV clone having a unique 3′ flanking sequence. Given the following description and teachings, one skilled in the art can apply the following methods to obtain any PERV clone described herein and is not intended to be limited to isolation of a specific PERV clone. A lambda library was constructed as described in Example 1. A PERV clone was isolated from a lambda FIX II vector using the PERV-A probe (SEQ ID NO:4). In order to distinguish a 3′ flanking sequence as distinct and unique (i.e. as a PERV locus), the PERV lambda clone was amplified using the primer pairs env5(2) and T3 or T7 (see
The G28-402A clone, containing a unique 3′ flanking sequence suitable for identification of potentially infectious PERV, was then subcloned into a pZerO-2 vector (Invitrogen, Calif.) at the vector's Not I site (
Once subcloned, G28-402A containing plasmids were transfected into human 293 cells (ATCC: CRL-1573) and positive transfectants obtained following G418 selection. Supernatant from the 293 cells containing a G28-402A clone was then co-cultured with hygromycin resistant 293 cells (Hygr) and grown under hygromycin selection.
PCR Analysis to Identify Allele Polymorphism of PERV Loci
A method to determine the existence of an allele containing or missing a specific PERV integration includes use of PCR analysis. Pig genomic DNA is analyzed with specific primers, which flank a particular PERV locus. Referring to
Referring to
Results of genotype analysis for allele polymorphism in a series of transgenic pigs is shown in the upper region of
Southern Blot Analysis to Identify Allele Polymorphism of PERV Loci
Another method to determine PERV loci allele polymorphism is by Southern blot analysis. Based upon the restriction map of each locus, a banding pattern can be predicted when the Southern blot is hybridized with a probe which recognizes each particular 3′ flanking genomic region. A different banding pattern will emerge if PERV is present depending upon whether PERV is present on both alleles, on one allele, or on neither allele. The noted polymorphism could be due to sequence polymorphism at the site of the particular restriction enzyme used, or to PERV integration genetic polymorphism. This can be tested by using multiple restriction enzymes. An example of a Southern blot analysis used to identify the various allele polymorphisms for a specific PERV locus is shown in
Southern Blot Analysis Conditions:
Porcine genomic DNA was digested with various restriction enzymes, size-fractionated on agarose gels, and transferred to nylon membranes. After cross-linking, the membranes were pre-hybridized in hybridization buffer (1% bovine serum albumin, 1 mM EDTA, 0.5 M NaHPO4, pH 7.2, 7% sodium dodeyl sulfate) for 2 hours at 65° C. Then fresh hybridization buffer containing 32P-labeled PERV probe was added and incubated overnight at 65° C. Probes were labeled with (32P) dCTP using T7 QuickPrime (Pharmacia, Piscataway, N.J.) After hybridization, nylon membranes were washed at 65° C. once in 2×SSC/0.2% SDS, once in 0.2×SSC/0.2% SDS, and subjected to autoradiography overnight.
Restriction digest with HindIII will result in a 1.3 kb fragment when probed with the G3–25, 3′flanking probe (SEQ ID NO:8) if a PERV-G3 allele is present in the sample. A sample not containing a PERV-G3 allele will result in a 3 kb size fragment.
Results from a Southern blot analysis of the PERV G19 locus are shown in
Results from PCR and Southern blot analysis of the PERV G28 locus are shown in
These methods allow screening of individual pigs to identify pigs that have one or both alleles without a specific PERV integration, making it possible to breed pigs in order to generate offspring which do not contain PERV loci that have the potential to be infectious.
As indicated in the examples above, Southern blot and PCR analysis demonstrated that all three PERV loci exhibit allele polymorphisms. Therefore, the present invention allows for breeding pigs that are negative for G3, G19 and G28 PERV loci.
Deposit of Material
The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, Va., 20110-2209, USA (ATCC):
These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of the deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Nextran, Inc. and ATCC which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
The present invention is not to be limited in scope by the construct(s) deposited, since the deposited embodiment(s) is/are intended as single illustration(s) of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material(s) herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
This application contains reference to numerous publications and patents, each of which is hereby incorporated, by reference in their entirety.
The present application claims priority to U.S. Ser. No. 60/279,337, filed Mar. 28, 2001, herein incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6100034 | Stoye et al. | Aug 2000 | A |
| 6190861 | Fishman | Feb 2001 | B1 |
| 6261806 | Banerjee et al. | Jul 2001 | B1 |
| 6867347 | Patience | Mar 2005 | B1 |
| Number | Date | Country |
|---|---|---|
| WO 0073512 | Dec 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20030224350 A1 | Dec 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60279337 | Mar 2001 | US |
