362
REPORTERGENES
[31]
antibody-containing solution (50-100/zl) should be placed on the medium, to one side of the tissue site, and the antibody allowed to diffuse outward. The protein/antibody reaction should be detectable as a white precipitate. Other Antibodies. In theory, the assay can be modified for use in screening for other reporter genes by using the antibodies specific for the encoded proteins, such as heat shock, chloramphenicol acyltransferase, or any other stable solution protein that can be induced and synthesized in the cytosol under the conditions of the culture. Concluding Remarks
Precautions. A positive reaction is not necessarily indicative of transformation. As with the standard GUS assay, the fluorescent product, 4-MU, can result from endogenous/3-glucuronidases of bacterial and fungal contaminants as well as enzymes endogenous to the plant tissue being assayed. 8 Fluorescent spots in culture medium can indicate contamination of the tissues with the Agrobacterium vector. Presence of a substrate such as MUG can induce synthesis of/3-glucuronidases in many saprophytic microorganisms in overnight assays. When this type of contamination is possible, the MUG assay buffer should contain an inhibitor of prokaryotic protein synthesis, such as chloramphenicol (170-200/zg/ml) or kanamycin (50-60 ~g/ml). 9 8 Cy Hu, P. Chee, R. Chesney, J. P. Miller, and W. O'Brien, Plant Cell Rep. 9, 1 (1990). 9 j. Sambrook, E. Fritsch, and T. Maniatis, "Molecular Cloning," 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
[31] S e c r e t e d P l a c e n t a l A l k a l i n e P h o s p h a t a s e as a Eukaryotic Reporter Gene
By BRYAN R. CULLEN and MICHAEL H. MALIM Introduction
A major focus of current molecular biological research is the identification and functional dissection of the cis-acting sequences and trans-acting factors that regulate eukaryotic gene expression in vivo. A common method of addressing this question is to link the regulatory sequence of interest, e.g., an inducible promoter/enhancer element, to a reporter gene. After transfection into the appropriate cells, the reporter gene product can then provide an accurate, quantitative measure of the level of gene METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[31]
SEAP REPORTERGENE
363
expression directed by the promoter/enhancer element, and mutated derivatives thereof, in response to wild-type or mutant forms of the appropriate trans-activator. The most common indicator gene assay relies on the prokaryotic gene for chloramphenicol acetyltransferase (CAT). ~ Chloramphenicol acetyltransferase has many advantages as a reporter gene, including the absence of interfering activities in eukaryotic cells, high protein stability, and high sensitivity. However, the most commonly utilized assay for CAT activity requires the use of an expensive radioactive substrate and is relatively time consuming. 1 In addition, cell cultures expressing the intracellular CAT enzyme must be harvested, lysed, and extracted prior to analysis. This precludes any kinetic analysis of gene expression within one culture over time and renders more difficult the simultaneous analysis of CAT RNA expression. These considerations have led to a search for alternate reporter genes that, ideally, would combine the sensitivity of the CAT assay with a more convenient, nonradioactive assay and that would produce a secreted indicator gene product. This quest has been given further impetus by the general perception that experiments designed to quantitate the level of gene expression in vivo require the use of an accurate internal control. Such a control must, by definition, rely on a second, distinct reporter gene construct that would not interfere with the accurate analysis of CAT activity. Several alternate or complementary reporter gene systems have been proposed. Among the more prevalent are the genes for the intracellular enzymes/3-galactosidase2 and luciferase3 and the secreted protein human growth hormone. 4 In this chapter, we will describe the use of a novel indicator gene, producing a secreted form of the human enzyme placental alkaline phosphase,5 that we believe offers a number of advantages. These include very high stability, efficient secretion by all cells tested, and the availability of a simple, inexpensive, and highly quantitative assay that does not require any unusual equipment or reagents. Principle of Method The gene for human placental alkaline phosphatase is a member of a multigene family that, in humans, includes at least three distinct iso1 C. M. Gorman, L. F. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982). 2 G. An, K. Hidaka, and L. Siminovitch, Mol. Cell. Biol. 2, 1628 (1982). 3 S. J. Gould and S. Subramani, Anal. Biochem. 7, 5 (1988). 4 R. F. Selden, K. Burke-Howie, M. E. Rowe, H. M. Goodman, and D. D. Moore, Mol. Cell. Biol. 6, 3173 (1986). 5 j. Berger, J. Hauber, R. Hauber, R. Geiger, and B. R. Cullen, Gene 66, 1 (1988).
364
REPORTER GENES Bgl X
EcoRV
[31]
Sal T Hind 111" 81)h T (AUG)
( 1903 bp)
pXF3 (220e bp ampR
pBC12/PL/SEAP (5225 bp)
~
~
L
Termination .~! ~Codon (1530 biD)
PvuI - " Rat InsulinGene (971 bp) Spliceend Poly(A) FIG. l. Structure of the plasmid pBC12/PL/SEAP. The plasmid is designed to facilitate insertion of promoter sequences upstream of the SEAP coding sequences. 5 The polylinker SphI site also encodes the translation initiation codon o f S E A P (GCAUGC). 6 Unique restriction enzyme sites are indicated.
zymes. 6'7 Although all alkaline phosphatases are able to hydrolyze phosphate esters, most optimally at high pH, placental alkaline phosphatase is unusual in at least two respects. The first is the high temperature stability of this enzyme relative to other isozymes detected in humans or in other animals. The second is the highly restricted species and tissue expression of the placental isozyme, which is confined to the placenta of higher primates and, occasionally, transformed cells derived from these same species. 6'7 Although alkaline phosphatases are normally membrane-bound cell surface proteins, they can be inefficiently released into supernatant media in an active form. 6-8 To convert the 513-amino acid cell surface form of placental alkaline phosphatase into a fully active, constitutively secreted enzyme, we introduced a termination codon after codon 489 to produce an efficiently secreted gene product that we term SEAP (secreted alkaline phosphatase). 5'8 The gene encoding SEAP was then introduced into a convenient shuttle vector termed pBC12/PL/SEAP. This plasmid (Fig. 1) contains the SEAP gene flanked 5' by a polylinker sequence and 6 W. Kam, E. Clauser, Y. S. Kim, Y. Wai Kan, and W. J. Rutter, Proc. Natl. Acad. Sci. U.S.A. 82, 8715 (1985). 7 j. L. Millan, J. Biol. Chem. 261, 3112 (1986). 8 j. Berger, A. D. Howard, L. Brink, L. Gerber, J. Hauber, B. R. Cullen, and S. Udenfriend, J. Biol. Chem. 263, 10016 (1988).
[31]
SEAP REPORTERGENE
365
3' by an intron and polyadenylation signals derived from the genomic rat preproinsulin II gene. 5 The ability to propagate the vector in bacteria is conferred by a selectable marker (Amp r) and origin of replication derived from the pBR322 derivative pXF3. In addition, pBC12/PL/SEAP contains a functional but transcriptionally inert origin of replication from simian virus 40 (SV40), thus permitting plasmid replication in cells that express functional SV40 T antigen. The polylinker sequence is convenient for the introduction of regulatory sequences of interest and derivatives containing the long terminal repeat (LTR) promoters of Rous sarcoma virus (RSV), human immunodeficiency virus (HIV), and human T cell leukemia virus (HTLV) have been described, as has a construct bearing the immediate early promoter of human cytomegalovirus. 5 After insertion of the promoter of interest into pBCI2/PL/SEAP, the expression plasmid can be introduced into tissue culture cell lines using an appropriate transfection protocol (the SV40 T antigen-expressing cell line COS is particularly ideal as this cell line is readily transfectable and can replicate the pBC12 series plasmids to high copy number). Vectors expressing trans-acting factors that modulate the activity of the introduced promoter can be cotransfected if desired. The SEAP enzyme is efficiently secreted into the supernatant medium in all cell lines tested thus far. Maximal levels are generally secreted between 48 and 72 hr after transfection. In our hands, SEAP activity is essentially stable in tissue culture media at 37°. Materials and Reagents Diethanolamine (Cat. No. D45-500; Fisher Scientific, Springfield, N J) L-Homoarginine and p-nitrophenol phosphate disodium (Cat. Nos. H-1007 and 104-0, respectively; Sigma Chemical Co., St. Louis, MO) 2 x SEAP buffer (for 50 ml):
Amount
Stock
Final concentration
10.51 g 50/xl 226 mg
Diethanolamine (100% solution) 1 M MgCI 2 L-Homoarginine
2M 1 mM 20 mM
Make up in water and store at 4° (no need to autoclave) 120 mMp-Nitrophenol phosphate: Dissolve 158 mg in 5 ml of 1 x SEAP buffer (make fresh)
366
REPORTER GENES
[31]
Assay for Secreted Alkaline Phosphatase 1. Transfect cells as normal and include a negative control (e.g., a transfection cocktail with no S E A P expression vector in it). 2. Culture at 37 ° for 48 to 72 hr. 3. R e m o v e 250 tzl o f each culture supernatant and transfer to Eppendoff tubes. To be on the safe side, maintain the cultures at 37 ° until " g o o d " S E A P data have been obtained. 4. Heat tubes at 65 ° for 5 min to inactivate endogenous phosphatases (SEAP is relatively heat stable). 5. Spin for 2 min at room temperature in an E p p e n d o r f microfuge at full speed. 6. Transfer supernatants to flesh E p p e n d o r f tubes. These may be stored at - 2 0 ° indefinitely. 7. In E p p e n d o r f tubes add 100 tzl of 2 × S E A P buffer to 100 tzl of the supernatant medium (or empirically determined dilutions thereof). As a zero standard make one sample substituting the medium with water. 8. Mix by vortexing. 9. Transfer the contents o f each tube to a well of a flat-bottomed microtiter plate. Avoid creating air bubbles. 10. Incubate the plate at 37 ° for I0 min. 11. During this incubation make the p-nitrophenol phosphate (substrate) solution and prewarm to 37 °. 12. Add 20/zl of the substrate solution to each well, preferably using a multipipetter. 13. Using an enzyme-linked immunosorbent assay (ELISA) plate reader, measure the light absorbance at 405 nm (A405) at regular intervals (e.g., e v e r y 5 min) o v e r the next 30 min while continuing to incubate the plate at 37 ° . 14. Calculate the levels of S E A P activity using data points that yield a rate of change in light absorbance that is linear with respect to time. (If available, a computer-linked kinetic E L I S A plate reader is most convenient, as this will precisely calculate the linear change in A4os in all 96 wells of a microtiter plate o v e r time.) 15. Secreted alkaline phosphatase activity can be expressed simply as the change in light absorbance per minute per sample. More formally, S E A P activity can be given in milliunits (mU) per milliliter, where 1 m U is defined as the amount of S E A P that will hydrolyze 1.0 pmol of pnitrophenyl phosphate per minute. 5 This equals an increase of 0.04 A405 units/min. The specific activity of S E A P is 2000 mU//zg protein. 9 9 E. Ezra, R. Blacher, and S. Udenfriend, Biochem. Biophys. Res. Commun. 116, 1076 (1983).
[31]
S E A P REPORTERGENE
367
2.0
2.0
A 1.8-
1.8-
1.6-
1.6-
1.4
1.4 -
1.2
1.2 -
1.0 0
8~
I0-
0.8
0.8 -
0.6
0.6 -
0.4
0.4-
02
0.2|
s
!
|
10 20
!
|
40
oo
TIME (min)
lb 2'0
i0
g0
TIME (rain)
FIG. 2. Analysis of in viuo promoter activity using the SEAP reporter gene. COS cell cultures were transfected 5 with the indicated expression plasmids and supernatant medium
sampled 60 hr later. The change in A405with time is shown for SEAP assays using either 10 /xl (A) or 100 /zl (B) of medium from transfected cultures. The assay was performed as described in text. The actual levels of SEAP activity determined in this assay were as follow: pBCI2/RSV/SEAP (A) = 275 mU/ml; pBCI2/H1V/SEAP (Tat induced) (11) = 247 mU/ml; pBC 12/HIV/SEAP (basal) (F1) = 2.2 mU/ml; negativecontrol (©) ~ 0.1 mU/ml. (Reproduced from Berger et al. 5 by permission.)
An example of an experiment in which the S E A P assay was used to quantitate relative promoter activities in transfected COS cell cultures is given in Fig. 2. In this experiment, we used plasmids that contained S E A P linked to the RSV LTR, a highly active constitutive promoter, or the H I V LTR, an inducible p r o m o t e r that displays a relatively low basal activity. These constructs were transfected into COS cell cultures either alone or, in the case of the p B C 1 2 / H I V / S E A P construct, together with an expression vector that encodes the H I V Tat trans-activator. 5 Supernatant media were sampled 60 hr after transfection and S E A P activity quantitated as described above. The figure presents the rate of change in light absorbance at 405 nm in S E A P assays that used either 10/zl (Fig. 2A) or 100 p.1 (Fig. 2B) of supernatant medium. The former level may be more suitable for accurate quantitative analysis of S E A P activity when very active promoter constructs are tested, in this case the RSV L T R and the trans-activated H I V LTR, while the latter may be more suitable for weak promoter constructs, such as the basal H I V L T R promoter. As demonstrated by
368
REPORTERGENES
[31]
this example, the SEAP assay is not only rapid and convenient but also sensitive and highly quantitative. In addition, this experiment demonstrates that endogenous alkaline phosphatases found either in tissue culture cells or fetal calf serum are not normally detected by this assay. This results both from the presence of L-homoarginine (an inhibitor of other alkaline phosphatases) in the assay buffer and from the preincubation at 65 °, which eliminates heat-sensitive isozymes. ~° Concluding Remarks In this chapter, we have described SEAP, a novel indicator gene producing secreted alkaline phosphatase. The SEAP gene has several advantages when compared to CAT or to other prevalent reporter genes. Notably, SEAP is a highly stable, secreted enzyme that can be accurately quantitated using an inexpensive and rapid assay that is similar to the alkaline phosphatase-based ELISA run routinely by many laboratories. ~j The fact that SEAP is secreted means that several assays can be performed on a single culture over time, thus permitting analysis of temporal changes in gene expression that occur in response to different stimuli. The SEAP assay can be used with the majority of cell types and has been shown to function extremely well in injected Xenopus oocytes. ~2 However, the SEAP assay does have one disadvantage relative to CAT, i.e., lower sensitivity. The assay described in this chapter cannot reliably quantitate levels of SEAP enzyme expression that fall significantly below 50 pg of protein per milliliter. This level of sensitivity is between 10- and 50-fold lower than the sensitivity of radioactivity-based assays for CAT expression.~ In part, this lower sensitivity may reflect a requirement for protein dimerization in the production of enzymatically active alkaline phosphatase. Therefore, in cells that are poorly transfectable or in other experimental settings where expression of the transfected plasmid is likely to be low, SEAP may not be suitable. Nevertheless, it is clear that SEAP will present considerable advantages in some settings and SEAP may well form an ideal internal control for CAT assays in many experimental protocols. The SEAP vectors described in this chapter are available from the authors on request.
10 j. Berger, A. D. Howard, L. Gerber, B. R. Cullen, and S. Udenfriend, Proc. Natl. A c a d . Sci. U . S . A . 84, 4885 (1987). ii R. B. McComb and G. N. Bowers, Jr., Clin. Chem. 18, 97 (1972). i2 S. S. Tate, R. Urade, R. Micanovic, L. Gerber, and S. Udenfriend, F A S E B J. 4, 227 (1990).
REPORTERGENES
[31]
antibody-containing solution (50-100/zl) should be placed on the medium, to one side of the tissue site, and the antibody allowed to diffuse outward. The protein/antibody reaction should be detectable as a white precipitate. Other Antibodies. In theory, the assay can be modified for use in screening for other reporter genes by using the antibodies specific for the encoded proteins, such as heat shock, chloramphenicol acyltransferase, or any other stable solution protein that can be induced and synthesized in the cytosol under the conditions of the culture. Concluding Remarks
Precautions. A positive reaction is not necessarily indicative of transformation. As with the standard GUS assay, the fluorescent product, 4-MU, can result from endogenous/3-glucuronidases of bacterial and fungal contaminants as well as enzymes endogenous to the plant tissue being assayed. 8 Fluorescent spots in culture medium can indicate contamination of the tissues with the Agrobacterium vector. Presence of a substrate such as MUG can induce synthesis of/3-glucuronidases in many saprophytic microorganisms in overnight assays. When this type of contamination is possible, the MUG assay buffer should contain an inhibitor of prokaryotic protein synthesis, such as chloramphenicol (170-200/zg/ml) or kanamycin (50-60 ~g/ml). 9 8 Cy Hu, P. Chee, R. Chesney, J. P. Miller, and W. O'Brien, Plant Cell Rep. 9, 1 (1990). 9 j. Sambrook, E. Fritsch, and T. Maniatis, "Molecular Cloning," 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
[31] S e c r e t e d P l a c e n t a l A l k a l i n e P h o s p h a t a s e as a Eukaryotic Reporter Gene
By BRYAN R. CULLEN and MICHAEL H. MALIM Introduction
A major focus of current molecular biological research is the identification and functional dissection of the cis-acting sequences and trans-acting factors that regulate eukaryotic gene expression in vivo. A common method of addressing this question is to link the regulatory sequence of interest, e.g., an inducible promoter/enhancer element, to a reporter gene. After transfection into the appropriate cells, the reporter gene product can then provide an accurate, quantitative measure of the level of gene METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[31]
SEAP REPORTERGENE
363
expression directed by the promoter/enhancer element, and mutated derivatives thereof, in response to wild-type or mutant forms of the appropriate trans-activator. The most common indicator gene assay relies on the prokaryotic gene for chloramphenicol acetyltransferase (CAT). ~ Chloramphenicol acetyltransferase has many advantages as a reporter gene, including the absence of interfering activities in eukaryotic cells, high protein stability, and high sensitivity. However, the most commonly utilized assay for CAT activity requires the use of an expensive radioactive substrate and is relatively time consuming. 1 In addition, cell cultures expressing the intracellular CAT enzyme must be harvested, lysed, and extracted prior to analysis. This precludes any kinetic analysis of gene expression within one culture over time and renders more difficult the simultaneous analysis of CAT RNA expression. These considerations have led to a search for alternate reporter genes that, ideally, would combine the sensitivity of the CAT assay with a more convenient, nonradioactive assay and that would produce a secreted indicator gene product. This quest has been given further impetus by the general perception that experiments designed to quantitate the level of gene expression in vivo require the use of an accurate internal control. Such a control must, by definition, rely on a second, distinct reporter gene construct that would not interfere with the accurate analysis of CAT activity. Several alternate or complementary reporter gene systems have been proposed. Among the more prevalent are the genes for the intracellular enzymes/3-galactosidase2 and luciferase3 and the secreted protein human growth hormone. 4 In this chapter, we will describe the use of a novel indicator gene, producing a secreted form of the human enzyme placental alkaline phosphase,5 that we believe offers a number of advantages. These include very high stability, efficient secretion by all cells tested, and the availability of a simple, inexpensive, and highly quantitative assay that does not require any unusual equipment or reagents. Principle of Method The gene for human placental alkaline phosphatase is a member of a multigene family that, in humans, includes at least three distinct iso1 C. M. Gorman, L. F. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982). 2 G. An, K. Hidaka, and L. Siminovitch, Mol. Cell. Biol. 2, 1628 (1982). 3 S. J. Gould and S. Subramani, Anal. Biochem. 7, 5 (1988). 4 R. F. Selden, K. Burke-Howie, M. E. Rowe, H. M. Goodman, and D. D. Moore, Mol. Cell. Biol. 6, 3173 (1986). 5 j. Berger, J. Hauber, R. Hauber, R. Geiger, and B. R. Cullen, Gene 66, 1 (1988).
364
REPORTER GENES Bgl X
EcoRV
[31]
Sal T Hind 111" 81)h T (AUG)
( 1903 bp)
pXF3 (220e bp ampR
pBC12/PL/SEAP (5225 bp)
~
~
L
Termination .~! ~Codon (1530 biD)
PvuI - " Rat InsulinGene (971 bp) Spliceend Poly(A) FIG. l. Structure of the plasmid pBC12/PL/SEAP. The plasmid is designed to facilitate insertion of promoter sequences upstream of the SEAP coding sequences. 5 The polylinker SphI site also encodes the translation initiation codon o f S E A P (GCAUGC). 6 Unique restriction enzyme sites are indicated.
zymes. 6'7 Although all alkaline phosphatases are able to hydrolyze phosphate esters, most optimally at high pH, placental alkaline phosphatase is unusual in at least two respects. The first is the high temperature stability of this enzyme relative to other isozymes detected in humans or in other animals. The second is the highly restricted species and tissue expression of the placental isozyme, which is confined to the placenta of higher primates and, occasionally, transformed cells derived from these same species. 6'7 Although alkaline phosphatases are normally membrane-bound cell surface proteins, they can be inefficiently released into supernatant media in an active form. 6-8 To convert the 513-amino acid cell surface form of placental alkaline phosphatase into a fully active, constitutively secreted enzyme, we introduced a termination codon after codon 489 to produce an efficiently secreted gene product that we term SEAP (secreted alkaline phosphatase). 5'8 The gene encoding SEAP was then introduced into a convenient shuttle vector termed pBC12/PL/SEAP. This plasmid (Fig. 1) contains the SEAP gene flanked 5' by a polylinker sequence and 6 W. Kam, E. Clauser, Y. S. Kim, Y. Wai Kan, and W. J. Rutter, Proc. Natl. Acad. Sci. U.S.A. 82, 8715 (1985). 7 j. L. Millan, J. Biol. Chem. 261, 3112 (1986). 8 j. Berger, A. D. Howard, L. Brink, L. Gerber, J. Hauber, B. R. Cullen, and S. Udenfriend, J. Biol. Chem. 263, 10016 (1988).
[31]
SEAP REPORTERGENE
365
3' by an intron and polyadenylation signals derived from the genomic rat preproinsulin II gene. 5 The ability to propagate the vector in bacteria is conferred by a selectable marker (Amp r) and origin of replication derived from the pBR322 derivative pXF3. In addition, pBC12/PL/SEAP contains a functional but transcriptionally inert origin of replication from simian virus 40 (SV40), thus permitting plasmid replication in cells that express functional SV40 T antigen. The polylinker sequence is convenient for the introduction of regulatory sequences of interest and derivatives containing the long terminal repeat (LTR) promoters of Rous sarcoma virus (RSV), human immunodeficiency virus (HIV), and human T cell leukemia virus (HTLV) have been described, as has a construct bearing the immediate early promoter of human cytomegalovirus. 5 After insertion of the promoter of interest into pBCI2/PL/SEAP, the expression plasmid can be introduced into tissue culture cell lines using an appropriate transfection protocol (the SV40 T antigen-expressing cell line COS is particularly ideal as this cell line is readily transfectable and can replicate the pBC12 series plasmids to high copy number). Vectors expressing trans-acting factors that modulate the activity of the introduced promoter can be cotransfected if desired. The SEAP enzyme is efficiently secreted into the supernatant medium in all cell lines tested thus far. Maximal levels are generally secreted between 48 and 72 hr after transfection. In our hands, SEAP activity is essentially stable in tissue culture media at 37°. Materials and Reagents Diethanolamine (Cat. No. D45-500; Fisher Scientific, Springfield, N J) L-Homoarginine and p-nitrophenol phosphate disodium (Cat. Nos. H-1007 and 104-0, respectively; Sigma Chemical Co., St. Louis, MO) 2 x SEAP buffer (for 50 ml):
Amount
Stock
Final concentration
10.51 g 50/xl 226 mg
Diethanolamine (100% solution) 1 M MgCI 2 L-Homoarginine
2M 1 mM 20 mM
Make up in water and store at 4° (no need to autoclave) 120 mMp-Nitrophenol phosphate: Dissolve 158 mg in 5 ml of 1 x SEAP buffer (make fresh)
366
REPORTER GENES
[31]
Assay for Secreted Alkaline Phosphatase 1. Transfect cells as normal and include a negative control (e.g., a transfection cocktail with no S E A P expression vector in it). 2. Culture at 37 ° for 48 to 72 hr. 3. R e m o v e 250 tzl o f each culture supernatant and transfer to Eppendoff tubes. To be on the safe side, maintain the cultures at 37 ° until " g o o d " S E A P data have been obtained. 4. Heat tubes at 65 ° for 5 min to inactivate endogenous phosphatases (SEAP is relatively heat stable). 5. Spin for 2 min at room temperature in an E p p e n d o r f microfuge at full speed. 6. Transfer supernatants to flesh E p p e n d o r f tubes. These may be stored at - 2 0 ° indefinitely. 7. In E p p e n d o r f tubes add 100 tzl of 2 × S E A P buffer to 100 tzl of the supernatant medium (or empirically determined dilutions thereof). As a zero standard make one sample substituting the medium with water. 8. Mix by vortexing. 9. Transfer the contents o f each tube to a well of a flat-bottomed microtiter plate. Avoid creating air bubbles. 10. Incubate the plate at 37 ° for I0 min. 11. During this incubation make the p-nitrophenol phosphate (substrate) solution and prewarm to 37 °. 12. Add 20/zl of the substrate solution to each well, preferably using a multipipetter. 13. Using an enzyme-linked immunosorbent assay (ELISA) plate reader, measure the light absorbance at 405 nm (A405) at regular intervals (e.g., e v e r y 5 min) o v e r the next 30 min while continuing to incubate the plate at 37 ° . 14. Calculate the levels of S E A P activity using data points that yield a rate of change in light absorbance that is linear with respect to time. (If available, a computer-linked kinetic E L I S A plate reader is most convenient, as this will precisely calculate the linear change in A4os in all 96 wells of a microtiter plate o v e r time.) 15. Secreted alkaline phosphatase activity can be expressed simply as the change in light absorbance per minute per sample. More formally, S E A P activity can be given in milliunits (mU) per milliliter, where 1 m U is defined as the amount of S E A P that will hydrolyze 1.0 pmol of pnitrophenyl phosphate per minute. 5 This equals an increase of 0.04 A405 units/min. The specific activity of S E A P is 2000 mU//zg protein. 9 9 E. Ezra, R. Blacher, and S. Udenfriend, Biochem. Biophys. Res. Commun. 116, 1076 (1983).
[31]
S E A P REPORTERGENE
367
2.0
2.0
A 1.8-
1.8-
1.6-
1.6-
1.4
1.4 -
1.2
1.2 -
1.0 0
8~
I0-
0.8
0.8 -
0.6
0.6 -
0.4
0.4-
02
0.2|
s
!
|
10 20
!
|
40
oo
TIME (min)
lb 2'0
i0
g0
TIME (rain)
FIG. 2. Analysis of in viuo promoter activity using the SEAP reporter gene. COS cell cultures were transfected 5 with the indicated expression plasmids and supernatant medium
sampled 60 hr later. The change in A405with time is shown for SEAP assays using either 10 /xl (A) or 100 /zl (B) of medium from transfected cultures. The assay was performed as described in text. The actual levels of SEAP activity determined in this assay were as follow: pBCI2/RSV/SEAP (A) = 275 mU/ml; pBCI2/H1V/SEAP (Tat induced) (11) = 247 mU/ml; pBC 12/HIV/SEAP (basal) (F1) = 2.2 mU/ml; negativecontrol (©) ~ 0.1 mU/ml. (Reproduced from Berger et al. 5 by permission.)
An example of an experiment in which the S E A P assay was used to quantitate relative promoter activities in transfected COS cell cultures is given in Fig. 2. In this experiment, we used plasmids that contained S E A P linked to the RSV LTR, a highly active constitutive promoter, or the H I V LTR, an inducible p r o m o t e r that displays a relatively low basal activity. These constructs were transfected into COS cell cultures either alone or, in the case of the p B C 1 2 / H I V / S E A P construct, together with an expression vector that encodes the H I V Tat trans-activator. 5 Supernatant media were sampled 60 hr after transfection and S E A P activity quantitated as described above. The figure presents the rate of change in light absorbance at 405 nm in S E A P assays that used either 10/zl (Fig. 2A) or 100 p.1 (Fig. 2B) of supernatant medium. The former level may be more suitable for accurate quantitative analysis of S E A P activity when very active promoter constructs are tested, in this case the RSV L T R and the trans-activated H I V LTR, while the latter may be more suitable for weak promoter constructs, such as the basal H I V L T R promoter. As demonstrated by
368
REPORTERGENES
[31]
this example, the SEAP assay is not only rapid and convenient but also sensitive and highly quantitative. In addition, this experiment demonstrates that endogenous alkaline phosphatases found either in tissue culture cells or fetal calf serum are not normally detected by this assay. This results both from the presence of L-homoarginine (an inhibitor of other alkaline phosphatases) in the assay buffer and from the preincubation at 65 °, which eliminates heat-sensitive isozymes. ~° Concluding Remarks In this chapter, we have described SEAP, a novel indicator gene producing secreted alkaline phosphatase. The SEAP gene has several advantages when compared to CAT or to other prevalent reporter genes. Notably, SEAP is a highly stable, secreted enzyme that can be accurately quantitated using an inexpensive and rapid assay that is similar to the alkaline phosphatase-based ELISA run routinely by many laboratories. ~j The fact that SEAP is secreted means that several assays can be performed on a single culture over time, thus permitting analysis of temporal changes in gene expression that occur in response to different stimuli. The SEAP assay can be used with the majority of cell types and has been shown to function extremely well in injected Xenopus oocytes. ~2 However, the SEAP assay does have one disadvantage relative to CAT, i.e., lower sensitivity. The assay described in this chapter cannot reliably quantitate levels of SEAP enzyme expression that fall significantly below 50 pg of protein per milliliter. This level of sensitivity is between 10- and 50-fold lower than the sensitivity of radioactivity-based assays for CAT expression.~ In part, this lower sensitivity may reflect a requirement for protein dimerization in the production of enzymatically active alkaline phosphatase. Therefore, in cells that are poorly transfectable or in other experimental settings where expression of the transfected plasmid is likely to be low, SEAP may not be suitable. Nevertheless, it is clear that SEAP will present considerable advantages in some settings and SEAP may well form an ideal internal control for CAT assays in many experimental protocols. The SEAP vectors described in this chapter are available from the authors on request.
10 j. Berger, A. D. Howard, L. Gerber, B. R. Cullen, and S. Udenfriend, Proc. Natl. A c a d . Sci. U . S . A . 84, 4885 (1987). ii R. B. McComb and G. N. Bowers, Jr., Clin. Chem. 18, 97 (1972). i2 S. S. Tate, R. Urade, R. Micanovic, L. Gerber, and S. Udenfriend, F A S E B J. 4, 227 (1990).
