- Email: [email protected]
A Mammalian Gene Expression Vector with Blue–White Selection for Efficient Subcloning inEscherichia coli
264
NOTES & TIPS
FIG. 1. Western blot of E. coli whole-cell lysates prepared with different protocols. E. coli cells (1 ml of saturating culture) harboring a mutant of aspartate aminotransferase (AspAT) were lysed with 100 ml Mg/SDS lysis buffer or 100 ml Laemmli sample buffer. Samples of 5 ml were fractionated with SDS–PAGE (10%) and electrotransferred to a nitrocellulose membrane. The blot was developed with antiAspAT antiserum, an alkaline phosphatase-labeled second antibody, and 5-bromo-4-chloro-3-indolyl phosphate (Sigma) plus nitrotetrazolium blue chloride (Fluka). Lane 1, sample prepared with Mg/ SDS lysis buffer and incubated 2 min at 1007C. Lane 2, sample prepared with Mg/SDS lysis buffer without heating. Lane 3, sample prepared with Laemmli sample buffer and incubated 2 min at 1007C (the sample prepared with Laemmli sample buffer without heating was unpipettable). The arrow indicates a degradation band in the heated samples.
somal DNA (1). DNase has also been used for the same purpose. Alternatively, exposure of the jelly-like SDS lysates to 1007C for 1 to 2 min will precipitate the DNA and thus reduce the viscosity. However, heating may cause the formation of breakdown product of target proteins. Here we describe a new method with which chromosomal DNA is easily removed without heating. In E. coli cells, the chromosomal DNA is in a compact form called nucleoid. When the cells lyse, their DNA spills out into long strands making the medium highly viscous. However, it was observed that the nucleoids remained condensed and the lysate was not viscous when the cells were lysed by mechanic means in the presence of Mg 2/ in high concentration (§1 M) (2). We therefore explored the feasibility of including MgCl2 in SDS lysis buffer for preparing whole-cell lysates and found the following composition of lysis buffer most effective in lysing the cells and precipitating chromosomal DNA, as well as denaturing proteins and dissociating protein complexes: Mg/SDS lysis buffer. Buffer consists of 0.1 M MgCl2 , 4% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue, and 100 mM Tris chloride, pH 6.8. Mg/SDS lysis procedure. (i) Spin 1 ml of a saturated E. coli culture (about 109 cells per milliliter) in a microfuge for 45 s. (ii) Add 100 ml of Mg/SDS lysis buffer to the cell pellet and vortex for 1 min to resuspend and lyse the cells. (50 ml or less Mg/SDS lysis buffer may be used to obtain more concentrated lysates for the detection of low-expression target proteins). (iii) Spin 2 min in a microfuge to remove the precipitate. The clear supernatant can now be used for SDS–PAGE. Although heating is equally effective in removing
DNA, it can cause the formation of breakdown products of some proteins (Fig. 1). Compared with sonication or repeated passage through a hypodermic needle, the Mg/SDS lysis procedure is more efficient in reducing viscosity and much simpler to perform. The presence of 0.1 M magnesium chloride in the samples does not alter the electrophoretic behavior of the proteins. To avoid the preparation and storage of two different sorts of sample buffers, the Mg/SDS lysis buffer can be used in place of Laemmli sample buffer (3) for soluble protein samples as well. In short, cell lysates devoid of chromosomal DNA and free of heating artifacts can be obtained with extreme ease with the Mg/SDS lysis procedure. REFERENCES 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Neidhardt, F. C., Ingraham, J. L., and Schaechter, M. (1990) Physiology of the Bacterial Cell. A Molecular Approach, Sinauer, Sunderland, MA. 3. Laemmli, U. K. (1970) Nature 227, 680–685.
A Mammalian Gene Expression Vector with Blue–White Selection for Efficient Subcloning in Escherichia coli Zhong Liu,* Linda M. Cashion,* Li Zhu,† and Ying Luo†,1 *Berlex Biosciences, 15049 San Pablo Avenue, P.O. Box 4099, Richmond, California 94804; and †CLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, California 94303-4230 Received November 1, 1996
Gene cloning is one of the essential steps in the study of protein structure and function. The pUC series of plasmids are the most widely used for this purpose because of their reliable and convenient blue–white selection feature in Escherichia coli (1). These types of vectors, however, have limited value in mammalian cells due to the lack of a mammalian transcription unit. With rapid progress of the human genome project, hundreds of thousands of new cDNA sequences have been cloned (2). A significant portion of these cDNA fragments will be transferred to eukaryotic expression vectors for subsequent structural and functional analysis. 1
To whom correspondence should be addressed. Fax: 415-3540776. ANALYTICAL BIOCHEMISTRY 246, 264–267 (1997) ARTICLE NO. AB972033
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
02-26-97 14:49:43
abnt
NOTES & TIPS
265
Transfer of genes into eukaryotic gene expression vectors sometimes involves multiple steps of plasmid manipulation in E. coli, which are not always very efficient. The aim of this work was to incorporate a blue– white selection feature (LacZ gene fragment) into a mammalian expression vector for easy subcloning in E. coli. This will facilitate the moving of cDNA fragments, especially at large scale, from regular cloning plasmids to eukaryotic expression vectors. Materials and Methods Construction of pCBW. An EcoRI–SapI fragment containing the CMV early promoter, full-length LacZ coding region, and SV40 polyadenylation signal was cloned into the AatII–SapI gap of pUC19. By selecting white colonies, we obtained the construct pCMVbD. An SalI–SapI deletion was made to remove the residual LacZ promoter to construct plasmid pCMVbD1. A useless unique EcoRI site in pCMVbD1 was destroyed by filling-in with Klenow to generate plasmid pCMVbD2. Two oligonucleotides were used to amplify by PCR the LacZ a fragment including the promoter, multiple cloning site (MCS),2 coding region, and terminator. The 5* splice site of the human b-globin first intron, including 24 bp upstream and 26 bp downstream, was synthesized into the upstream oligo. The PCR product was then inserted into the BspEI–NotI gap of pCMVbD2. By selecting blue colonies, pCMVbD3 was obtained. To insert a 3* splice site into the MCS of LacZ a fragment in-frame, we synthesized three oligos. Annealing of these three oligos together formed a DNA fragment containing the 3* splice site including 57 bp upstream (branch point is located at 37 bp upstream) and 25 bp downstream. An ‘‘A’’ 24 bp upstream of the 3* splice site was converted to ‘‘C’’ to destroy the stop codon. This DNA fragment was then cloned into the EcoRI– HindIII gap of pCMVbD3. The new construct was named pCMVbD4. Finally, an AatII–SmaI fragment was blunt-ended by Klenow and replaced the ClaI– KpnI fragment of pCMVbD4. The final construct was named pCBW. CAT assay. CAT expression was measured in transient transfection assays. An equal molar amount of CAT reporter constructs (total DNA 10 mg) was introduced into CHO-K1 cells by a calcium phosphate transfection protocol (5). pGl3-control vector (0.2 mg) (Promega, Cat. No. E1741) was also included in each transfection as a control. Total cell lysates were prepared 48 h after transfection. Levels of CAT production were measured by ELISA (a kit from BoehringerMannheim, Cat. No. 1 363 727). The luciferase activity from the transfection control vector was measured in light units of luminescence with a luciferase assay sys2
Abbreviations used: MCS, multiple cloning site.
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FIG. 1. Schematic diagram of pCBW and pCBW-CAT. (A) The orientation of LacZ fragment is in reverse orientation to the direction of CMV early promoter. A 3* splice site is immediately upstream of the multiple cloning site. The direction of E. coli promoter driving LacZ expression is indicated. ‘‘T’’ represents the termination site of LacZ transcription. After splicing as indicated, the majority of LacZ sequence including its terminator region will be removed. (B) A CAT gene is inserted into the multiple cloning site of pCBW. E. coli colonies transformed by pCBW-CAT will not turn blue in the presence of X-Gal. The sizes of the RNA probe and protected fragments in RNase protection assay are also shown.
tem (Promega, Cat. No. E4030). The relative CAT expression was obtained by normalizing the amount of CAT (ng) against luciferase activity (arbitrary light units). During transfection, the appropriate amount of pBluescript was supplemented to each transfection mixture to ensure a total of 10 mg DNA per 10-cm tissue culture plate. Results and Discussion To achieve high levels of gene expression in mammalian cells, the CMV early promoter was chosen to construct pCBW. The replication origin from pUC19 was used to obtain high copy number in E. coli. The LacZ a fragment containing its promoter, coding sequence, and terminator from pUC19 was placed downstream of the CMV promoter in a reverse orientation so that the LacZ gene will not be expressed in mammalian cells. In addition, ATG of LacZ in this design will not interfere with translation of the inserted gene (Fig. 1A).
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FIG. 2. CAT assay result. All CAT assays were done 48 h after transfection. Values of the relative CAT activity are shown on the top of each bar. The final CAT expression levels are presented by normalizing CAT against luciferase activity. Numbers represent the averages of two sets of independent experiments.
brought closer to the 5* end of the transcript for efficient translation initiation. The only LacZ sequence still remaining in the 3* untranslated region of the mRNA transcript is the E. coli promoter plus the translation start site of LacZ. A CAT gene was amplified by PCR and cloned into the XhoI–HindIII site of pCBW. In a blue–white selection of ligated product, 97% of the colonies turned white on X-Gal/Amp plates, while the pUC19 transformants remained blue. No significant difference in the speed to turn blue or the intensity of the blue color was observed between pCBW and pUC19 transformants. Ten white colonies were selected for restriction enzyme analysis. All of them contained inserts of expected size. Two of these clones were amplified for transfection into mammalian cells. CAT expression was measured by transient transfection. Two CAT expression vectors driven by CMV promoters, pcDNA I/Amp-CAT and pCI-neo-CAT, were used in control experiments. Equal molar amount of pCBW-CAT, pcDNA I/Amp-CAT, and pCI-neo-CAT reporter plasmids were introduced into CHO-K1 cells by a calcium phosphate transfection protocol in a balanced transfection (3). To correct for the difference in transfection efficiencies, a fixed amount of a luciferase expression vector was also cotransfected. Our results indicate that pCBW-based CAT gene expression is ú50-fold higher than pcDNA I/Amp-CAT and 2-fold higher than pCI-neo-CAT (Fig. 2). The de-
Since RNA processing is known to play a central role in the stability and accumulation of mRNAs (4–9), an efficient intron from human b-globin was included in the transcription unit of pCBW. The 5* splice site of the first intron of b-globin was inserted between the transcription initiation site of the CMV promoter and the transcription termination site of LacZ. The 3* splice site of the first intron, including the branch point and a polypyrimidine tract, was then inserted in-frame into the multiple cloning sites of LacZ. A stop codon was mutated by changing A at position 27 bp upstream of the 3* splice site to C. An SV40 polyadenylation site was placed downstream of the LacZ promoter for efficient termination and polyadenylation of the primary transcripts in mammalian cells. A cDNA fragment inserted into the MCS interrupts the open reading frame of the LacZ gene and prevents the bacteria from turning blue in the presence of X-Gal. After being transfected into mammalian cells, however, the intron containing most of the LacZ sequence will be removed by splicing so that the inserted gene is
FIG. 3. RNase protection assay of pCBW-CAT transcript. Protected riboprobes were resolved on the 6% denaturing polyacrylamide gel. Approximately 1 mg of poly(A)/ mRNA was used in each reaction, supplemented with 20 mg of total RNA from untransfected CHOK1 cells. RNase protection assay was performed using a RPA II kit (Ambion, Cat. No. 1410). Bands representing spliced and unspliced transcripts are indicated. Lane 1, undigested riboprobe; lane 2, endlabeled DNA size marker derived from a 100-bp ladder from Life Technologies; lane 3, CHO-K1 cells transfected with pCBW; lane 4, CHO-K1 cells transfected with pCBW-CAT.
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sign of the blue–white selection in our vector apparently does not interfere with the expression of cDNA inserted into the MCS. To verify the efficient removal of LacZ sequences by splicing in mammalian cells, we performed an RNase protection assay on poly(A)/ RNA from CHO-K1 cells transfected with pCBW-CAT. Forty-eight hours after transfection, poly(A)/ mRNA samples were prepared for analysis. The results from an RNase protection assay indicate that only about 1% of the messages was unspliced (Fig. 3). This suggests that the 3* splice site upstream of the polylinker region is efficiently recognized by mammalian splicing machinery and the LacZ sequence inside the intron is absent from the final transcript. In our design, splicing in mammalian cells removes the nonessential LacZ sequences. Therefore, unpredictable effects by these unnecessary sequences on target gene expression are reduced to a minimum level (10, 11). The blue–white selection design can be incorporated into most mammalian gene expression vectors as a standard feature in the future. The use of splicing to remove unnecessary elements also points to a new direction for construction of eukaryotic expression vectors. Although regulatory sequences of gene expression in prokaryotic and eukaryotic organisms are different, by placing yeast or E. coli promoters in the intron in the same orientation with a mammalian promoter, genes inserted into the MCS can be efficiently transcribed in E. coli, yeast, and mammalian cells. Together with a
previous finding that a mutant 5* UTR can fulfill the requirements of both prokaryotic and eukaryotic translation machinery, construction of a different kind of shuttle vectors for gene expression in E. coli, yeast, and mammalian cells is plausible (12).
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Acknowledgments. We thank Jenifer Fishel for her critical reading of the manuscript.
REFERENCES 1. Vieira, J., and Messing, J. (1982) Gene 19, 259–268. 2. Adams, M. D., Kerlavage, A. R., Fleischmann, R. D., Fuldner, R. A., Bult, C. J., Lee, N. H., Kirkness, E. F., Weinstock, K. G., Gocayne, J. D., White, O., et al. (1995) Nature 377(Suppl.), 3– 174. 3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995) Current Protocols in Molecular Biology, Wiley, New York. 4. Barret, N. L., Carmichael, G. G., and Luo, Y. (1994) Nucleic Acids Res. 19, 3011–3017. 5. Buchman, A. R., and Berg, P. (1988) Mol Cell Biol. 8, 4395– 4405. 6. Huang, M. T. F., and Gorman, C. M. (1990) Nucleic Acids Res. 18, 937–947. 7. McKeown, M. (1992) Annu. Rev. Cell Biol. 8, 135–155. 8. Neuberger, M. S., and Williams, G. T. (1988) Nucleic Acids Res. 166, 713–724. 9. Rio, D. C. (1992) Curr. Opin. Cell Biol. 4, 444–4522. 10. Manzella, J. M., and Blackshear, P. J. (1990) J. Biol. Chem. 265, 11817–11822. 11. Kauppinen, L. (1995) FEBS Lett. 365, 61–65. 12. Al-Qahtani, A., and Mensa-Wilmot, K. (1996) Nucleic Acids Res. 24, 1173–1174.
NOTES & TIPS
FIG. 1. Western blot of E. coli whole-cell lysates prepared with different protocols. E. coli cells (1 ml of saturating culture) harboring a mutant of aspartate aminotransferase (AspAT) were lysed with 100 ml Mg/SDS lysis buffer or 100 ml Laemmli sample buffer. Samples of 5 ml were fractionated with SDS–PAGE (10%) and electrotransferred to a nitrocellulose membrane. The blot was developed with antiAspAT antiserum, an alkaline phosphatase-labeled second antibody, and 5-bromo-4-chloro-3-indolyl phosphate (Sigma) plus nitrotetrazolium blue chloride (Fluka). Lane 1, sample prepared with Mg/ SDS lysis buffer and incubated 2 min at 1007C. Lane 2, sample prepared with Mg/SDS lysis buffer without heating. Lane 3, sample prepared with Laemmli sample buffer and incubated 2 min at 1007C (the sample prepared with Laemmli sample buffer without heating was unpipettable). The arrow indicates a degradation band in the heated samples.
somal DNA (1). DNase has also been used for the same purpose. Alternatively, exposure of the jelly-like SDS lysates to 1007C for 1 to 2 min will precipitate the DNA and thus reduce the viscosity. However, heating may cause the formation of breakdown product of target proteins. Here we describe a new method with which chromosomal DNA is easily removed without heating. In E. coli cells, the chromosomal DNA is in a compact form called nucleoid. When the cells lyse, their DNA spills out into long strands making the medium highly viscous. However, it was observed that the nucleoids remained condensed and the lysate was not viscous when the cells were lysed by mechanic means in the presence of Mg 2/ in high concentration (§1 M) (2). We therefore explored the feasibility of including MgCl2 in SDS lysis buffer for preparing whole-cell lysates and found the following composition of lysis buffer most effective in lysing the cells and precipitating chromosomal DNA, as well as denaturing proteins and dissociating protein complexes: Mg/SDS lysis buffer. Buffer consists of 0.1 M MgCl2 , 4% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue, and 100 mM Tris chloride, pH 6.8. Mg/SDS lysis procedure. (i) Spin 1 ml of a saturated E. coli culture (about 109 cells per milliliter) in a microfuge for 45 s. (ii) Add 100 ml of Mg/SDS lysis buffer to the cell pellet and vortex for 1 min to resuspend and lyse the cells. (50 ml or less Mg/SDS lysis buffer may be used to obtain more concentrated lysates for the detection of low-expression target proteins). (iii) Spin 2 min in a microfuge to remove the precipitate. The clear supernatant can now be used for SDS–PAGE. Although heating is equally effective in removing
DNA, it can cause the formation of breakdown products of some proteins (Fig. 1). Compared with sonication or repeated passage through a hypodermic needle, the Mg/SDS lysis procedure is more efficient in reducing viscosity and much simpler to perform. The presence of 0.1 M magnesium chloride in the samples does not alter the electrophoretic behavior of the proteins. To avoid the preparation and storage of two different sorts of sample buffers, the Mg/SDS lysis buffer can be used in place of Laemmli sample buffer (3) for soluble protein samples as well. In short, cell lysates devoid of chromosomal DNA and free of heating artifacts can be obtained with extreme ease with the Mg/SDS lysis procedure. REFERENCES 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Neidhardt, F. C., Ingraham, J. L., and Schaechter, M. (1990) Physiology of the Bacterial Cell. A Molecular Approach, Sinauer, Sunderland, MA. 3. Laemmli, U. K. (1970) Nature 227, 680–685.
A Mammalian Gene Expression Vector with Blue–White Selection for Efficient Subcloning in Escherichia coli Zhong Liu,* Linda M. Cashion,* Li Zhu,† and Ying Luo†,1 *Berlex Biosciences, 15049 San Pablo Avenue, P.O. Box 4099, Richmond, California 94804; and †CLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, California 94303-4230 Received November 1, 1996
Gene cloning is one of the essential steps in the study of protein structure and function. The pUC series of plasmids are the most widely used for this purpose because of their reliable and convenient blue–white selection feature in Escherichia coli (1). These types of vectors, however, have limited value in mammalian cells due to the lack of a mammalian transcription unit. With rapid progress of the human genome project, hundreds of thousands of new cDNA sequences have been cloned (2). A significant portion of these cDNA fragments will be transferred to eukaryotic expression vectors for subsequent structural and functional analysis. 1
To whom correspondence should be addressed. Fax: 415-3540776. ANALYTICAL BIOCHEMISTRY 246, 264–267 (1997) ARTICLE NO. AB972033
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
02-26-97 14:49:43
abnt
NOTES & TIPS
265
Transfer of genes into eukaryotic gene expression vectors sometimes involves multiple steps of plasmid manipulation in E. coli, which are not always very efficient. The aim of this work was to incorporate a blue– white selection feature (LacZ gene fragment) into a mammalian expression vector for easy subcloning in E. coli. This will facilitate the moving of cDNA fragments, especially at large scale, from regular cloning plasmids to eukaryotic expression vectors. Materials and Methods Construction of pCBW. An EcoRI–SapI fragment containing the CMV early promoter, full-length LacZ coding region, and SV40 polyadenylation signal was cloned into the AatII–SapI gap of pUC19. By selecting white colonies, we obtained the construct pCMVbD. An SalI–SapI deletion was made to remove the residual LacZ promoter to construct plasmid pCMVbD1. A useless unique EcoRI site in pCMVbD1 was destroyed by filling-in with Klenow to generate plasmid pCMVbD2. Two oligonucleotides were used to amplify by PCR the LacZ a fragment including the promoter, multiple cloning site (MCS),2 coding region, and terminator. The 5* splice site of the human b-globin first intron, including 24 bp upstream and 26 bp downstream, was synthesized into the upstream oligo. The PCR product was then inserted into the BspEI–NotI gap of pCMVbD2. By selecting blue colonies, pCMVbD3 was obtained. To insert a 3* splice site into the MCS of LacZ a fragment in-frame, we synthesized three oligos. Annealing of these three oligos together formed a DNA fragment containing the 3* splice site including 57 bp upstream (branch point is located at 37 bp upstream) and 25 bp downstream. An ‘‘A’’ 24 bp upstream of the 3* splice site was converted to ‘‘C’’ to destroy the stop codon. This DNA fragment was then cloned into the EcoRI– HindIII gap of pCMVbD3. The new construct was named pCMVbD4. Finally, an AatII–SmaI fragment was blunt-ended by Klenow and replaced the ClaI– KpnI fragment of pCMVbD4. The final construct was named pCBW. CAT assay. CAT expression was measured in transient transfection assays. An equal molar amount of CAT reporter constructs (total DNA 10 mg) was introduced into CHO-K1 cells by a calcium phosphate transfection protocol (5). pGl3-control vector (0.2 mg) (Promega, Cat. No. E1741) was also included in each transfection as a control. Total cell lysates were prepared 48 h after transfection. Levels of CAT production were measured by ELISA (a kit from BoehringerMannheim, Cat. No. 1 363 727). The luciferase activity from the transfection control vector was measured in light units of luminescence with a luciferase assay sys2
Abbreviations used: MCS, multiple cloning site.
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FIG. 1. Schematic diagram of pCBW and pCBW-CAT. (A) The orientation of LacZ fragment is in reverse orientation to the direction of CMV early promoter. A 3* splice site is immediately upstream of the multiple cloning site. The direction of E. coli promoter driving LacZ expression is indicated. ‘‘T’’ represents the termination site of LacZ transcription. After splicing as indicated, the majority of LacZ sequence including its terminator region will be removed. (B) A CAT gene is inserted into the multiple cloning site of pCBW. E. coli colonies transformed by pCBW-CAT will not turn blue in the presence of X-Gal. The sizes of the RNA probe and protected fragments in RNase protection assay are also shown.
tem (Promega, Cat. No. E4030). The relative CAT expression was obtained by normalizing the amount of CAT (ng) against luciferase activity (arbitrary light units). During transfection, the appropriate amount of pBluescript was supplemented to each transfection mixture to ensure a total of 10 mg DNA per 10-cm tissue culture plate. Results and Discussion To achieve high levels of gene expression in mammalian cells, the CMV early promoter was chosen to construct pCBW. The replication origin from pUC19 was used to obtain high copy number in E. coli. The LacZ a fragment containing its promoter, coding sequence, and terminator from pUC19 was placed downstream of the CMV promoter in a reverse orientation so that the LacZ gene will not be expressed in mammalian cells. In addition, ATG of LacZ in this design will not interfere with translation of the inserted gene (Fig. 1A).
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266
NOTES & TIPS
FIG. 2. CAT assay result. All CAT assays were done 48 h after transfection. Values of the relative CAT activity are shown on the top of each bar. The final CAT expression levels are presented by normalizing CAT against luciferase activity. Numbers represent the averages of two sets of independent experiments.
brought closer to the 5* end of the transcript for efficient translation initiation. The only LacZ sequence still remaining in the 3* untranslated region of the mRNA transcript is the E. coli promoter plus the translation start site of LacZ. A CAT gene was amplified by PCR and cloned into the XhoI–HindIII site of pCBW. In a blue–white selection of ligated product, 97% of the colonies turned white on X-Gal/Amp plates, while the pUC19 transformants remained blue. No significant difference in the speed to turn blue or the intensity of the blue color was observed between pCBW and pUC19 transformants. Ten white colonies were selected for restriction enzyme analysis. All of them contained inserts of expected size. Two of these clones were amplified for transfection into mammalian cells. CAT expression was measured by transient transfection. Two CAT expression vectors driven by CMV promoters, pcDNA I/Amp-CAT and pCI-neo-CAT, were used in control experiments. Equal molar amount of pCBW-CAT, pcDNA I/Amp-CAT, and pCI-neo-CAT reporter plasmids were introduced into CHO-K1 cells by a calcium phosphate transfection protocol in a balanced transfection (3). To correct for the difference in transfection efficiencies, a fixed amount of a luciferase expression vector was also cotransfected. Our results indicate that pCBW-based CAT gene expression is ú50-fold higher than pcDNA I/Amp-CAT and 2-fold higher than pCI-neo-CAT (Fig. 2). The de-
Since RNA processing is known to play a central role in the stability and accumulation of mRNAs (4–9), an efficient intron from human b-globin was included in the transcription unit of pCBW. The 5* splice site of the first intron of b-globin was inserted between the transcription initiation site of the CMV promoter and the transcription termination site of LacZ. The 3* splice site of the first intron, including the branch point and a polypyrimidine tract, was then inserted in-frame into the multiple cloning sites of LacZ. A stop codon was mutated by changing A at position 27 bp upstream of the 3* splice site to C. An SV40 polyadenylation site was placed downstream of the LacZ promoter for efficient termination and polyadenylation of the primary transcripts in mammalian cells. A cDNA fragment inserted into the MCS interrupts the open reading frame of the LacZ gene and prevents the bacteria from turning blue in the presence of X-Gal. After being transfected into mammalian cells, however, the intron containing most of the LacZ sequence will be removed by splicing so that the inserted gene is
FIG. 3. RNase protection assay of pCBW-CAT transcript. Protected riboprobes were resolved on the 6% denaturing polyacrylamide gel. Approximately 1 mg of poly(A)/ mRNA was used in each reaction, supplemented with 20 mg of total RNA from untransfected CHOK1 cells. RNase protection assay was performed using a RPA II kit (Ambion, Cat. No. 1410). Bands representing spliced and unspliced transcripts are indicated. Lane 1, undigested riboprobe; lane 2, endlabeled DNA size marker derived from a 100-bp ladder from Life Technologies; lane 3, CHO-K1 cells transfected with pCBW; lane 4, CHO-K1 cells transfected with pCBW-CAT.
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sign of the blue–white selection in our vector apparently does not interfere with the expression of cDNA inserted into the MCS. To verify the efficient removal of LacZ sequences by splicing in mammalian cells, we performed an RNase protection assay on poly(A)/ RNA from CHO-K1 cells transfected with pCBW-CAT. Forty-eight hours after transfection, poly(A)/ mRNA samples were prepared for analysis. The results from an RNase protection assay indicate that only about 1% of the messages was unspliced (Fig. 3). This suggests that the 3* splice site upstream of the polylinker region is efficiently recognized by mammalian splicing machinery and the LacZ sequence inside the intron is absent from the final transcript. In our design, splicing in mammalian cells removes the nonessential LacZ sequences. Therefore, unpredictable effects by these unnecessary sequences on target gene expression are reduced to a minimum level (10, 11). The blue–white selection design can be incorporated into most mammalian gene expression vectors as a standard feature in the future. The use of splicing to remove unnecessary elements also points to a new direction for construction of eukaryotic expression vectors. Although regulatory sequences of gene expression in prokaryotic and eukaryotic organisms are different, by placing yeast or E. coli promoters in the intron in the same orientation with a mammalian promoter, genes inserted into the MCS can be efficiently transcribed in E. coli, yeast, and mammalian cells. Together with a
previous finding that a mutant 5* UTR can fulfill the requirements of both prokaryotic and eukaryotic translation machinery, construction of a different kind of shuttle vectors for gene expression in E. coli, yeast, and mammalian cells is plausible (12).
AID
abnt
AB $NAT
/
6m29$$$422
02-26-97 14:49:43
Acknowledgments. We thank Jenifer Fishel for her critical reading of the manuscript.
REFERENCES 1. Vieira, J., and Messing, J. (1982) Gene 19, 259–268. 2. Adams, M. D., Kerlavage, A. R., Fleischmann, R. D., Fuldner, R. A., Bult, C. J., Lee, N. H., Kirkness, E. F., Weinstock, K. G., Gocayne, J. D., White, O., et al. (1995) Nature 377(Suppl.), 3– 174. 3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995) Current Protocols in Molecular Biology, Wiley, New York. 4. Barret, N. L., Carmichael, G. G., and Luo, Y. (1994) Nucleic Acids Res. 19, 3011–3017. 5. Buchman, A. R., and Berg, P. (1988) Mol Cell Biol. 8, 4395– 4405. 6. Huang, M. T. F., and Gorman, C. M. (1990) Nucleic Acids Res. 18, 937–947. 7. McKeown, M. (1992) Annu. Rev. Cell Biol. 8, 135–155. 8. Neuberger, M. S., and Williams, G. T. (1988) Nucleic Acids Res. 166, 713–724. 9. Rio, D. C. (1992) Curr. Opin. Cell Biol. 4, 444–4522. 10. Manzella, J. M., and Blackshear, P. J. (1990) J. Biol. Chem. 265, 11817–11822. 11. Kauppinen, L. (1995) FEBS Lett. 365, 61–65. 12. Al-Qahtani, A., and Mensa-Wilmot, K. (1996) Nucleic Acids Res. 24, 1173–1174.