Abstract:
A method is disclosed herein of providing transgenic  Xenopus laevis  oocytes. The method comprises preparing transgenic  Xenopus laevis  oocytes at a first location and transporting the transgenic  Xenopus laevis  oocytes to a second location remote from the first location. Preferably, the transgenic  Xenopus laevis  oocytes are prepared by injecting  Xenopus laevis  oocytes with a cRNA or cDNA encoding various human or animal membrane proteins. In a further aspect of the subject invention, a culture system is provided which comprises at least one vessel and at least one transgenic  Xenopus laevis  oocyte disposed in the vessel. The vessel is sealed so as to allow for transportation of the vessel. Advantageously, with the subject invention, a user is able to conduct testing, e.g., drug transport assays, with transgenic  Xenopus laevis  oocytes without the arduous task of injecting  Xenopus laevis  oocytes with cRNA or cDNA encoding various human or animal membrane proteins.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]     This Application claims priority to U.S. Provisional Application No. 60/562,981, filed Apr. 19, 2004, the entire contents of which are incorporated by reference herein. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates to a method of providing  Xenopus laevis  oocytes which have been pre-injected with a cRNA or cDNA encoding human or animal membrane proteins, such as membrane transport proteins.  
       BACKGROUND OF THE INVENTION  
       [0003]     The oocyte from the South African clawed  Xenopus laevis  frog is an often used functional expression system. Oocyte expression systems have been used to study the function of membrane proteins such as transporters, ion channels, and pumps. The oocyte expression systems demonstrate low backgrounds, high expression levels and proper post-translational modifications.  
         [0004]     Setting up drug transport assays for various human and animal membrane transport proteins (also referred to herein as transporters), such as human Organic Cation Transporter 1 (hOCT1, SLC 22A1), human Organic Anion Transporting Polypeptide 1 (hOATP1, SLC21A3), human Organic Anion Transporting Polypeptide 2 (hOATP2, SLC21A6), human Organic Anion Transporting Polypeptide 8 (hOATP8, SLC21A8), human Na + -Taurocholate Cotransport Protein (hNTCP, SLC10A1), rat Organic Anion Transporting Polypeptide (rOatp1, Slc21a1), human Peptide Transporter 1 (hPEPT1, SLC15A1), human Peptide Transporter 2 (hPEPT2, SLC15A2), human Organic Anion Transporter 1 (hOAT1, SLC22A6), human Organic Anion Transporter 3 (hOAT3, SLC22A8), rat Organic Anion Transporter 3 (rOat3, Slc22a8), and rat Organic Anion Transporting Polypeptide 4 (rOatp4, Slc21a10) can present many technical challenges. Expressing these transporters in oocytes for their function characterizations is difficult and time consuming owing to the scale of the process.  
       SUMMARY OF THE INVENTION  
       [0005]     A method is disclosed herein of providing transgenic  Xenopus laevis  oocytes. The method comprises preparing transgenic  Xenopus laevis  oocytes at a first location and transporting the transgenic  Xenopus laevis  oocytes to a second location remote from the first location. Preferably, the transgenic  Xenopus laevis  oocytes are prepared by injecting  Xenopus laevis  oocytes with a cRNA or cDNA encoding various human or animal membrane proteins.  
         [0006]     In a further aspect of the subject invention, a culture system is provided which comprises at least one vessel and at least one transgenic  Xenopus laevis  oocyte disposed in the vessel. The vessel is sealed so as to allow for transportation of the vessel. Advantageously, with the subject invention, a user is able to conduct testing, e.g., drug transport assays, with transgenic  Xenopus laevis  oocytes without the arduous task of injecting  Xenopus laevis  oocytes with cRNA or cDNA encoding various human or animal membrane proteins.  
         [0007]     The cRNA or cDNA may encode various human or animal membrane proteins, such as human or animal membrane transport proteins selected from human Organic Cation Transporter 1 (hOCT1), human Organic Anion Transporting Polypeptide 1 (hOATP1), human Organic Anion Transporting Polypeptide 2 (hOATP2), human Organic Anion Transporting Polypeptide 8 (hOATP8), human Na + -Taurocholate Cotransport Protein (hNTCP), rat Organic Anion Transporting Polypeptide (rOatp1), human Peptide Transporter 1 (hPEPT1), human Peptide Transporter 2 (hPEPT2), human Organic Anion Transporter 1 (hOAT1), human Organic Anion Transporter 3 (hOAT3), rat Organic Anion Transporter 3 (rOat3), and rat Organic Anion Transporting Polypeptide 4 (rOatp4).  
         [0008]     As used herein, “transgenic” refers to oocytes that express a human or animal membrane protein, such as a membrane transport protein, in addition to the normal complement of proteins.  
         [0009]     These and other features of the invention will be better understood through a study of the following detailed description and accompanying drawings. 
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0010]      FIG. 1  is a flowchart representing the method of the subject invention.  
         [0011]      FIG. 2  is a cross-section of packaged transgenic and control  Xenopus laevis  oocytes. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0012]     With reference to  FIG. 1 , a method  10  is set forth of providing transgenic  Xenopus laevis  oocytes.  
         [0013]     In an initial step  12 , the transgenic  Xenopus laevis  oocytes are prepared. Any known technique can be used to prepare the transgenic  Xenopus laevis  oocytes. Preferably, the transgenic  Xenopus laevis  oocytes are prepared by injecting  Xenopus laevis  oocytes with cRNA or cDNA encoding a human or animal membrane protein, such as a membrane transport protein. The cRNA or cDNA may encode various human or animal membrane proteins, including membrane transport proteins. By way of non-limiting example, the cRNA or cDNA may encode any of the following animal or human membrane transport proteins: human Organic Cation Transporter 1 (hOCT1, SLC 22A1), human Organic Anion Transporting Polypeptide 1 (hOATP1, SLC21A3), human Organic Anion Transporting Polypeptide 2 (hOATP2, SLC21A6), human Organic Anion Transporting Polypeptide 8 (hOATP8, SLC21A8), human Na + -Taurocholate Cotransport Protein (hNTCP, SLC10A1), rat Organic Anion Transporting Polypeptide (rOatp1, Slc21a1), human Peptide Transporter 1 (hPEPT1, SLC15A1), human Peptide Transporter 2 (hPEPT2, SLC15A2), human Organic Anion Transporter 1 (hOAT1, SLC22A6), human Organic Anion Transporter 3 (hOAT3, SLC22A8), rat Organic Anion Transporter 3 (rOat3, Slc22a8), and rat Organic Anion Transporting Polypeptide 4 (rOatp4, Slc21a10). These proteins and the cRNA&#39;s and the cDNA&#39;s that encode them are known in the art.  
         [0014]     In a preferred embodiment,  Xenopus laevis  frogs or  Xenopus laevis  oocytes may be obtained from NASCO, Ft. Atkinson, Wis. 53538. The cRNA or cDNA encoding human or animal membrane proteins may be injected by standard techniques. See, Wagner, et al.,  Cellular Physiol. Biochem.,  2000, 10, 1-12. Also, conventional molecular biological or cell biological techniques that can be employed with the present invention are disclosed in  Current Protocols in Molecular Biology,  Volumes I-III (F. Ausubel, ed. 1994).  
         [0015]     Once the transgenic  Xenopus laevis  oocytes are prepared, the transgenic  Xenopus laevis  oocytes can be transported from the site of preparation to a remote site, as represented by step  14 . With the subject invention, the transgenic  Xenopus laevis  oocytes are prepared at a first location (e.g., a manufacturer&#39;s facility) and transported to a remote, second location (e.g., the customer&#39;s facility). Any mode of transportation can be used, including, but not limited to automobile, truck, airplane, train, or ship transport, or any combinations thereof. It is preferred that the transgenic  Xenopus laevis  oocytes be transported for a period of no more than 2-4 days. The viability of transgenic  Xenopus laevis  oocytes is typically a period of 7 days.  
         [0016]     Preferably, the transgenic  Xenopus laevis  oocytes are disposed in one or more vessels (step  16 ) prior to being transporting. The vessels may be of any known configuration, such as test tubes, vials, flasks, etc. With reference to  FIG. 2 , vessels  18  are preferably wells of a multiwell plate  20 . It is further preferred that the multiwell plate  20  conform to conventional multiwell plate standards (e.g., the Standards of the Society of Biomolecular Screening) so as to be usable in drug assay handling equipment (e.g., high throughput screening (HTS) equipment). The multiwell plate  20  is preferably the multiwell plate commercially referred to under the trademark “BD Falcon™ Flip-Lock Packaging” from Becton Dickinson &amp; Co., Franklin Lakes, N.J. This specific multiwell plate includes an array of twelve wells, although any number of wells can be used with the subject invention.  
         [0017]     With further reference to  FIG. 2 , any number of transgenic  Xenopus laevis  oocytes  22  may be disposed into the vessels  18 . It is preferred that 4-5 transgenic  Xenopus laevis  oocytes  22  be disposed per each of the vessels  18 .  
         [0018]     A buffer solution  24  is also preferably disposed into the vessels  18  to suspend the transgenic  Xenopus laevis  oocytes  22 . The buffer solution  24  may be any solution which will maintain the viability of the transgenic  Xenopus laevis  oocytes  22  for an extended period to allow for transportation to the remote site. The buffer solution  24  may be ND96 (96 mM NaCl, 2 mM KCl, 1 mM CL 2 , 1.8 mM CaCl 2 , 50 μg/ml Gentamicin, pH 7.4) or Modified Barth Medium (88 mM NaCl, 0.82 mM MgSO 4 , 0.41 mM CaCl 2 , 0.33 mM Ca(NO 3 ) 2 , 2.4 mM NaHCO 3 , 10 mM HEPES, 50 μg/ml Gentamicin). It is preferred that approximately 5 ml of the buffer solution  24  be provided to suspend 4-5 transgenic  Xenopus laevis  oocytes in each one of the vessels  18 . It is further preferred that with the vessels  18  being part of a unitary structure (e.g., wells of a single multiwell plate), each of the vessels  18  of the unitary structure include an equal amount of the buffer solution  24 , even in the vessels  18  where no transgenic  Xenopus laevis  oocytes  22  may be present. Equal amounts of the buffer solution  24  will reduce sloshing effects during transportation and reduce potential damage to the transgenic  Xenopus laevis  oocytes  22 .  
         [0019]     The buffer solution  24  may be disposed into the vessels  18  using any known technique. It is preferred to dispose the buffer solution  24  into the vessels  18  prior to disposing the transgenic  Xenopus laevis  oocytes  22 . The transgenic  Xenopus laevis  oocytes  22  may be disposed into the vessels  18  using any known technique, such as transfer pipette.  
         [0020]     It is also preferred that the vessels be sealed prior to transportation, as indicated by step  26  in  FIG. 1 . The vessels are preferably sealed liquid-tight to prevent leakage of the transgenic  Xenopus laevis  oocytes and the buffer solution. Any known sealing arrangement can be used. For illustrative purposes, and with reference to  FIG. 2 , a resilient gasket or seal member  28  (e.g., a silicone gasket) may be disposed across the vessel  18  to provide a liquid-tight barrier against leakage. The seal member  28  may be fixed relative to the vessel  18  using any known technique. For example, a lid  30  may be placed over the seal member  28  which is threaded, hinged, latched or otherwise removably fixed relative to the vessel  18  (e.g., being latched onto the multiwell plate  20 ). The lid  30  may provide a backing force against the seal member  28  to enhance the seal provided thereby. The seal member  28  may also be a foil or plastic film removably fixed relative to the vessel (e.g., by heat bonding).  
         [0021]     Transgenic  Xenopus laevis  oocytes are temperature sensitive, and it is preferred to maintain the temperature of the transgenic  Xenopus laevis  oocytes during the transportation step  14 . More particularly, it is preferred that the temperature be maintained in the range of 10-20° C., and, more preferably, at a temperature of 16° C. The temperature can be maintained by any known technique. With the subject invention, the transgenic  Xenopus laevis  oocytes may be initially chilled to the desired temperature and packaged in insulative material to maintain the chilled temperature. For example, with reference to  FIG. 2 , a paper sleeve or envelope  32  (e.g., heavy weight paper or paperboard) and/or a plastic sleeve or pouch  34  may be placed about the vessel  18  to provide insulation. The paper sleeve or envelope  32  and/or the plastic sleeve or pouch  34  may be tightly wrapped about the vessel  18  to additionally provide backing force to the seal member  28 . In addition, external insulative packaging may be used. With reference to  FIG. 2 , the vessel  18  may be packed into a carton  36  having an insulative liner  38 . Loose packaging material  40  (e.g., foam segments) may also be provided to add not only insulation, but also shock-resistance. Further, pre-cooled media  42  may be packaged externally of the transgenic  Xenopus laevis  oocytes (e.g., pre-cooled gel packs chilled to 4° C.) to further maintain the desired temperature. To allow for inspection of temperature fluctuations during transportation, a thermal digital recorder  44  may be packaged with the transgenic  Xenopus laevis  oocytes.  
         [0022]     As will be appreciated by those skilled in the art, the transgenic  Xenopus laevis  oocytes of the subject invention can be used as part of a culture system. In addition to the transgenic  Xenopus laevis  oocytes being sealed in one or more vessels for transportation, the culture system may include control  Xenopus laevis  oocytes. With reference to  FIG. 1 , the control  Xenopus laevis  oocytes may be prepared by providing un-injected  Xenopus laevis  oocytes (i.e., not injected with a cRNA or cDNA encoding a human or animal membrane protein) or by injecting water into the  Xenopus laevis  oocytes in vivo (step  46 ). Thereafter, the control  Xenopus laevis  oocytes may be disposed into one or more vessels (step  48 ), sealed (step  26 ) and transported (step  14 ), including maintaining the temperature thereof, in the same manner as described above. With reference to  FIG. 2 , control  Xenopus laevis  oocytes  50  are shown. It is, however, preferred that a unitary structure of vessels (e.g., a multiwell plate) not contain both transgenic  Xenopus laevis  oocytes and control  Xenopus laevis  oocytes so as to avoid confusion; it is preferred that separated vessels be used.  
         [0023]     The culture system may also include other components, such as reagents for analysis. The reagents may include ND96 buffer solution, as described above, (e.g., 500 ml); sodium (Na + ) buffer solution (e.g., 500 ml); and, SDS lysis buffer solution (e.g., 30 ml).