Gene, 112 (1992) 257-260 © 1992Elsevier Science Publishers B.V. AI! fights reserved. 0378-1119/92]$05.00
257
GENE063~
Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay (Recombinant DNA; marker gene; reporter gene)
Michael SchandorfSmensen, Mogens Duch, Kirsten Paludan, Poul Jmgensen and Finn Skou Pedersen Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C (Denmark) Received by A.M. Skalka: 12 July 1991 Revised/Accepted: i October/7 October 1991 Received at publishers: 30 December 1991
SUMMARY
Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene nee; hph and nee may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.
INTRODUCTION
Prokaryotic antibiotic resistance genes have proved useful in a variety of experimental strategies involving transient or stable transfer of genetic material into mammalian cells. Among the genes encoding resistance against aminoglycoside antibiotics, nee has been widely used as a dominant selectable marker for mammalian cells (Colb6re-Garapin
Correspondenceto: Dr. F.S. Pedersen, Department of Molecular Biology, University of Aarhus, C.F. Moilers All6 130, DK-8000 Aarhus C (Denmark) Tel. ( + 45)86125177 or ( + 45)86202711-2262; Fax ( + 45)86196226. Abbreviations: Akv, Akv murine leukemia virus; bp, base pair(s); ddH20, double-distilled water; ELISA, enzyme-linked immunosorbent assay; G418, Geneticin; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; HPH, Hy phosphotransferase; hph, gene encoding HPH (HyR); Hy, hygromycin B; kb, kilobase(s) or 1000 bp; Km, kanamycin; LTR, long terminal repeat; nee, gene encoding NPT II (neomycin resistance); NPT II, neomycin phosphotransferase; PBS, phosphate-buffered saline (116 mM NaCl/3 mM KH2PO4/5.5 mM Na2HPO4); tv, transmission vector; ~-2, murine leukemia virus-packagingcell line (Mann et al., 1983).
et ai., 1981; Southern and Berg, 1982) and quantitative assays for NPT II enzymatic activity in mammalian cell extracts have been developed (Reiss et al., 1984; Platt and Yang, 1987; Duch et al., 1990). Another prokaryotic aminoglycoside antibiotic resistance gene, hph, encodes the enzyme HPH that inactivates Hy, which is toxic to mammalian cells via interference with protein synthesis (Gonzalez et al., 1978; Rao et al., 1983; Gritz and Davies, 1983). The dominant selectable marker hph can be used together with nee for experiments in higher eukaryotic cells that require two non-interfering selectable markers (Blochinger and Diggelmann, 1984; Hu and Temin, 1990). We are interested in using such a double marker system to study cis- and trans-determinants for expression of retroviral proviruses. For use in these studies we have generated parallel sets of murine leukemia virus derived retroviral vectors carrying nee (Paludan et al., 1989a,b) or hph (unpublished) and employed simple quantitative assays for their gene products. We here report on quantitative measurement of HPH activity in crude mammalian cell extracts by a simple dot-assay. The assay is adapted from the phosphocellulose binding assays for aminoglycoside modifying
258 enzymes in bacterial extracts (Haas and Dowding, 1975; Rao et al., 1983; Santerra et al., 1984) with the modifications introduced in the dot assays for NPT II in mammalian cell extracts (Platt and Yang, 1987; Duch et al., 1990). Our results point to the value of a combined use of neo and hph as a set of two non-interfering selectable marker/ reporter genes in mammalian cells.
EXPERIMENTAL AND DISCUSSION
(a) Transmission and expression of hph The retroviral vector tvAkv-hph (unpublished) used for transfer and expression of hph is homologous to previously described tvAkv-neo (Paludan et al., 1989a), except that neo was replaced by hph (derived from pSV2hyg, kindly provided by Dr. L. Gritz), inserted in the plus orientation under control of LTR-driven expression. DNA of a plasmid carrying tvAkv-hph was introduced into cultures of 0-2 packaging cells (Mann et al., 1983) by transfection, and L691 cells (McGrath et al., 1980) growing in suspension were subsequently infected by co-cultivation with the transfected 0-2 populations. Hy-resistant L691 cells could be derived from the co-cultivated L691 population by selection for growth in 600 #g Hy/ml, whereas no resistant cells could be obtained from uninfected cultures. Transfer and expression of hph was monitored by RNA dot-blot hybridization analysis using an hph-specific probe. Independently infected cultures of Hy-resistant cells were all found to express hph-RNA by dot-blot hybridization analysis, as shown in Fig. 1 for the culture selected for further study, whereas no hph-RNA signal was detected in uninfected L691 cells. This indicates that Hy-resistance must be due to production of HPH. (b) Quantitative determination of HPH activity in crude cell extracts Crude cell extracts of Hy-resistant L691 cells were used to establish a simple quantitative dot-assay for HPH according to the principles used in the previously described assays for NPT II (Platt and Yang, 1987; Duch et al., 1990), in which aliquots of cell extracts are incubated with [ ~-32p]ATP and the antibiotic substrate (Km) and the samples filtered through one layer of nitrocellulose and a double layer of phosphocellulose in a minifold dot-blot apparatus. The proteins, some of which may be radioactively labelled due to cellular protein kinases, are adsorbed to the nitrocellulose. The positively charged Km phosphate will bind to the phosphocellulose, whereas ATP will pass through. The amount of radioactivity in a dot on the phosphocellulose may be taken as a measure of NPT II activity. This assay was adapted for determination of HPH activity using Hy as the enzymatic substrate instead of Km. Since Hy is
GAPDH HPH i
i@i@
L691/Akv-hph
[//I [
Fig. i. Detection of hph-RNA in Hy-resistant L691 cells after infection with tvAkv-hph transmission vectors. The cell sample was a population of L691 cells selected for growth in 600 pg Hy/ml (Sigma Chemical Co., St. Louis, MO) after co-cultivation for three days with ~b-2cells transletted with vector plasmids as described previously (Paludan et al., 1989a). The L691 cells growing in suspension were separated from the adherent ~-2 cells by repeated transfers to new culture flasks. Total RNA purification was done as described by Chomczynski and Sacchi (1987). Crude RNA was transferred to Zeta-probe membranes (Bio-Rad Laboratories, Richmond, CA) using a minifold essentially as described by Sambrook et al. (1989). The probe for detection ofhph-RNA was a 1.06-kb fragment encompassing the complete HPH-encoding region. The probe for detection of GAPDH mRNA used as an internal standard was a 1.3.kb Pstl fragment derived from pRGADPH-13 (Fort etal., 1985). The DNA probes were 32p labeled to a specific activity of about 5 x l0 s dpm/~g essentially as described by Feinberg and Vogelstein (1983). Hybridization and washing were performed as described in the Zeta-probe instruction manual (Bio-Rad Laboratories). The autoradiogram shows duplicate determinations of a population infected with tvAkv-hph, and of uninfected L691 cells. The films were exposed at -80°C using intensifying screens. Exposure times were 5 h for the hph-probe and 16 h for the GADPHencoding probe.
only weakly positively charged at neutral pH (The Merck Index, 1989), care was taken to ensure electrostatic retention of Hy phosphate on the negatively charged phosphocellulose membrane. In our procedure, described in the legend to Fig. 2, this was accomplished by using low ionicstrength buffers during all transfer and washing steps. The series of HPH assays shown in Fig. 2 were performed using a crude extract of HPH expressing L691 cells after serial dilutions with a crude extract from non-infected L691 cells. The amount of radioactivity (Hy [32p]phosphate) on the phosphocellulose was determined by densitometry of an autoradiogram as described previously for the NPT II assay (Ouch et al., 1990). A linear relationship was observed between the amount of HPH-containing extract and absorbance over about two order s of magnitude. Other series of assays (not shown) have demonstrated that the assay can be used for HPH determination in a linear range up to about 10 #g of protein per sampie.
(c) Absence of cross-reactivity between HPH and NPT II assays Previous studies (Blochinger and Diggelmann, 1984; Hu and Temin, 1990) have demonstrated that neo and hph confer distinct resistance properties upon mammalian cells. We have confirmed those observations in that L691 cells
259
HPH assay
NPT II assay
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257
GENE063~
Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay (Recombinant DNA; marker gene; reporter gene)
Michael SchandorfSmensen, Mogens Duch, Kirsten Paludan, Poul Jmgensen and Finn Skou Pedersen Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C (Denmark) Received by A.M. Skalka: 12 July 1991 Revised/Accepted: i October/7 October 1991 Received at publishers: 30 December 1991
SUMMARY
Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene nee; hph and nee may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.
INTRODUCTION
Prokaryotic antibiotic resistance genes have proved useful in a variety of experimental strategies involving transient or stable transfer of genetic material into mammalian cells. Among the genes encoding resistance against aminoglycoside antibiotics, nee has been widely used as a dominant selectable marker for mammalian cells (Colb6re-Garapin
Correspondenceto: Dr. F.S. Pedersen, Department of Molecular Biology, University of Aarhus, C.F. Moilers All6 130, DK-8000 Aarhus C (Denmark) Tel. ( + 45)86125177 or ( + 45)86202711-2262; Fax ( + 45)86196226. Abbreviations: Akv, Akv murine leukemia virus; bp, base pair(s); ddH20, double-distilled water; ELISA, enzyme-linked immunosorbent assay; G418, Geneticin; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; HPH, Hy phosphotransferase; hph, gene encoding HPH (HyR); Hy, hygromycin B; kb, kilobase(s) or 1000 bp; Km, kanamycin; LTR, long terminal repeat; nee, gene encoding NPT II (neomycin resistance); NPT II, neomycin phosphotransferase; PBS, phosphate-buffered saline (116 mM NaCl/3 mM KH2PO4/5.5 mM Na2HPO4); tv, transmission vector; ~-2, murine leukemia virus-packagingcell line (Mann et al., 1983).
et ai., 1981; Southern and Berg, 1982) and quantitative assays for NPT II enzymatic activity in mammalian cell extracts have been developed (Reiss et al., 1984; Platt and Yang, 1987; Duch et al., 1990). Another prokaryotic aminoglycoside antibiotic resistance gene, hph, encodes the enzyme HPH that inactivates Hy, which is toxic to mammalian cells via interference with protein synthesis (Gonzalez et al., 1978; Rao et al., 1983; Gritz and Davies, 1983). The dominant selectable marker hph can be used together with nee for experiments in higher eukaryotic cells that require two non-interfering selectable markers (Blochinger and Diggelmann, 1984; Hu and Temin, 1990). We are interested in using such a double marker system to study cis- and trans-determinants for expression of retroviral proviruses. For use in these studies we have generated parallel sets of murine leukemia virus derived retroviral vectors carrying nee (Paludan et al., 1989a,b) or hph (unpublished) and employed simple quantitative assays for their gene products. We here report on quantitative measurement of HPH activity in crude mammalian cell extracts by a simple dot-assay. The assay is adapted from the phosphocellulose binding assays for aminoglycoside modifying
258 enzymes in bacterial extracts (Haas and Dowding, 1975; Rao et al., 1983; Santerra et al., 1984) with the modifications introduced in the dot assays for NPT II in mammalian cell extracts (Platt and Yang, 1987; Duch et al., 1990). Our results point to the value of a combined use of neo and hph as a set of two non-interfering selectable marker/ reporter genes in mammalian cells.
EXPERIMENTAL AND DISCUSSION
(a) Transmission and expression of hph The retroviral vector tvAkv-hph (unpublished) used for transfer and expression of hph is homologous to previously described tvAkv-neo (Paludan et al., 1989a), except that neo was replaced by hph (derived from pSV2hyg, kindly provided by Dr. L. Gritz), inserted in the plus orientation under control of LTR-driven expression. DNA of a plasmid carrying tvAkv-hph was introduced into cultures of 0-2 packaging cells (Mann et al., 1983) by transfection, and L691 cells (McGrath et al., 1980) growing in suspension were subsequently infected by co-cultivation with the transfected 0-2 populations. Hy-resistant L691 cells could be derived from the co-cultivated L691 population by selection for growth in 600 #g Hy/ml, whereas no resistant cells could be obtained from uninfected cultures. Transfer and expression of hph was monitored by RNA dot-blot hybridization analysis using an hph-specific probe. Independently infected cultures of Hy-resistant cells were all found to express hph-RNA by dot-blot hybridization analysis, as shown in Fig. 1 for the culture selected for further study, whereas no hph-RNA signal was detected in uninfected L691 cells. This indicates that Hy-resistance must be due to production of HPH. (b) Quantitative determination of HPH activity in crude cell extracts Crude cell extracts of Hy-resistant L691 cells were used to establish a simple quantitative dot-assay for HPH according to the principles used in the previously described assays for NPT II (Platt and Yang, 1987; Duch et al., 1990), in which aliquots of cell extracts are incubated with [ ~-32p]ATP and the antibiotic substrate (Km) and the samples filtered through one layer of nitrocellulose and a double layer of phosphocellulose in a minifold dot-blot apparatus. The proteins, some of which may be radioactively labelled due to cellular protein kinases, are adsorbed to the nitrocellulose. The positively charged Km phosphate will bind to the phosphocellulose, whereas ATP will pass through. The amount of radioactivity in a dot on the phosphocellulose may be taken as a measure of NPT II activity. This assay was adapted for determination of HPH activity using Hy as the enzymatic substrate instead of Km. Since Hy is
GAPDH HPH i
i@i@
L691/Akv-hph
[//I [
Fig. i. Detection of hph-RNA in Hy-resistant L691 cells after infection with tvAkv-hph transmission vectors. The cell sample was a population of L691 cells selected for growth in 600 pg Hy/ml (Sigma Chemical Co., St. Louis, MO) after co-cultivation for three days with ~b-2cells transletted with vector plasmids as described previously (Paludan et al., 1989a). The L691 cells growing in suspension were separated from the adherent ~-2 cells by repeated transfers to new culture flasks. Total RNA purification was done as described by Chomczynski and Sacchi (1987). Crude RNA was transferred to Zeta-probe membranes (Bio-Rad Laboratories, Richmond, CA) using a minifold essentially as described by Sambrook et al. (1989). The probe for detection ofhph-RNA was a 1.06-kb fragment encompassing the complete HPH-encoding region. The probe for detection of GAPDH mRNA used as an internal standard was a 1.3.kb Pstl fragment derived from pRGADPH-13 (Fort etal., 1985). The DNA probes were 32p labeled to a specific activity of about 5 x l0 s dpm/~g essentially as described by Feinberg and Vogelstein (1983). Hybridization and washing were performed as described in the Zeta-probe instruction manual (Bio-Rad Laboratories). The autoradiogram shows duplicate determinations of a population infected with tvAkv-hph, and of uninfected L691 cells. The films were exposed at -80°C using intensifying screens. Exposure times were 5 h for the hph-probe and 16 h for the GADPHencoding probe.
only weakly positively charged at neutral pH (The Merck Index, 1989), care was taken to ensure electrostatic retention of Hy phosphate on the negatively charged phosphocellulose membrane. In our procedure, described in the legend to Fig. 2, this was accomplished by using low ionicstrength buffers during all transfer and washing steps. The series of HPH assays shown in Fig. 2 were performed using a crude extract of HPH expressing L691 cells after serial dilutions with a crude extract from non-infected L691 cells. The amount of radioactivity (Hy [32p]phosphate) on the phosphocellulose was determined by densitometry of an autoradiogram as described previously for the NPT II assay (Ouch et al., 1990). A linear relationship was observed between the amount of HPH-containing extract and absorbance over about two order s of magnitude. Other series of assays (not shown) have demonstrated that the assay can be used for HPH determination in a linear range up to about 10 #g of protein per sampie.
(c) Absence of cross-reactivity between HPH and NPT II assays Previous studies (Blochinger and Diggelmann, 1984; Hu and Temin, 1990) have demonstrated that neo and hph confer distinct resistance properties upon mammalian cells. We have confirmed those observations in that L691 cells
259
HPH assay
NPT II assay
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