CA2203613C - Secreted .alpha.-amylase as a reporter gene - Google Patents
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- CA2203613C CA2203613C CA 2203613 CA2203613A CA2203613C CA 2203613 C CA2203613 C CA 2203613C CA 2203613 CA2203613 CA 2203613 CA 2203613 A CA2203613 A CA 2203613A CA 2203613 C CA2203613 C CA 2203613C
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- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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Abstract
The regulation of gene expression is of fundamental importance to all biological functions including adaptation to environmental conditions, cell division and differentiation, and the development of disease states such as cancer. The application of "reporter" gene systems, which allow changes in gene expression to be assayed quickly and easily, has contributed greatly to our understanding of the mechanisms involved in gene regulation. Reporter genes come in two basic varieties; those that encode intracellular enzymes, and those that code for secreted reporter proteins. Most reporter genes currently in use are of the intracellular variety that require manipulation of the cells, normally harvesting and lysis, prior to measurement of the reporter activity. In contrast, cells transformed with constructs that encode secreted reporter enzymes do not require manipulation or lysis of the cells for activity measurements.
Instead, aliquots of the extracellular medium are harvested and assayed directly for enzyme activity. Thus, secreted reporter genes can be used to monitor gene expression in vitro, in vivo and ex vivo. The present invention relates to a reporter gene encoding the chicken a-amylase enzyme for use in transforming mammalian cells. The use of a-amylase as a secreted reporter gene is particularly attractive because the enzyme is extremely stable over a wide range of conditions, measurement of .alpha.-amylase activity is simple, quantitative, sensitive, safe and inexpensive, and the range of electrophoretically-distinguishable variants available allows the assays to be performed in virtually any host without interference from endogenous amylase activities.
Instead, aliquots of the extracellular medium are harvested and assayed directly for enzyme activity. Thus, secreted reporter genes can be used to monitor gene expression in vitro, in vivo and ex vivo. The present invention relates to a reporter gene encoding the chicken a-amylase enzyme for use in transforming mammalian cells. The use of a-amylase as a secreted reporter gene is particularly attractive because the enzyme is extremely stable over a wide range of conditions, measurement of .alpha.-amylase activity is simple, quantitative, sensitive, safe and inexpensive, and the range of electrophoretically-distinguishable variants available allows the assays to be performed in virtually any host without interference from endogenous amylase activities.
Description
SECRETED a-AMYLASE AS A REPORTER GENE
The present invention relates to a secreted reporter gene. More specifically the present invention relates to a secreted reporter gene based on chicken a-amylase for use in mammalian cells.
BACKGROUND OF THE INVENTION
There are a number of commercially available reporter genes that encode intracellular enzymes, e.g. green fluorescent protein (GFP; Chalfie et al., 1994, Science 263: 802-805 ), luciferase (de Wet et al., 1987, Mol. Cell. Biol. 7:
725-737),' chloramphenicol acetyltransferase (CAT; Gorman et al., 1982, Mol. Cell. Biol.
The present invention relates to a secreted reporter gene. More specifically the present invention relates to a secreted reporter gene based on chicken a-amylase for use in mammalian cells.
BACKGROUND OF THE INVENTION
There are a number of commercially available reporter genes that encode intracellular enzymes, e.g. green fluorescent protein (GFP; Chalfie et al., 1994, Science 263: 802-805 ), luciferase (de Wet et al., 1987, Mol. Cell. Biol. 7:
725-737),' chloramphenicol acetyltransferase (CAT; Gorman et al., 1982, Mol. Cell. Biol.
2:
1044-1051). However, none of these intracellular enzymes is suitable as a secreted reporter activity. For instance, the luciferase reporter gene is used widely due mainly to the sensitivity of the detection system and the lack of background activity in most biological systems. However, the luciferase enzyme is relatively unstable under any conditions, and is completely and irreversibly inactivated by the process of secretion.
Secretable human placental alkaline phosphatase (SEAP; a truncated form of membrane-bound alkaline phosphatase; Kam et al., 1985, Proc. Natl. Acad. Sci.
USA
~. .
82: 8715-8719) is a reporter gene that is distributed by Clontech as a secreted reporte:r gene/enzyme system (see European Patent application 0 327 960). However, many biological systems display endogenous (background) alkaline phosphatase (AP) activity.
For example, the high level of alkaline phosphatase activity in cows' milk precludes the use of SEAP as a reporter activity for experiments aimed at using the bovine udder as a "bioreactor" in the production of bioengineered milk. A second commercially-available, secreted reporter gene is human growth hormone (hGH, Boehringer Mannheim; Selden et al., 1986, Mol. Cell. Biol. 6: 31.73-3179 ). However, since hGH:
has no easily measurable biological activity, the detection system for this secreted product uses a laborious, indirect antibody-based ELISA method.
* trademark Thus there is a need for an improved secreted reporter gene that overcomes the problems of the prior art systems. Specifically there is a need for a secreted reporter gene encoding a biological activity that, unlike hGH, is safe and easily measured, but that does not have the background activity problems associated with SEAP.
The chicken a-amylase reporter gene encodes a biological activity that is easily measured by a variety of liquid or semi-solid phase assay systems. The use of the preferred, native gel assay system eliminates any potential interference by endogenous amylase activities produced by the transformed, mammalian target cells. In addition, the advantage of using a secreted reporter activity is that it is not necessary to harvest cells from the culture dish, or to biopsy a transformed tissue (e.g. udder epithelium), nor is it necessary to lyse cells prior to assay. Instead, the cell culture supernatant or biological fluid (e.g. milk) is recovered from the transformed culture or animal and assayed for reporter activity. This feature has several advantages such as the ability to conduct multiple measurements over time on a single transformed population of cells (time course measurements).
The construction of the reporter gene involved the use of standard recombinant DNA methods, including the use of restriction enzymes to fuse DNA molecules witli coherent ends, the amplification of DNA fragments using the polymerase chain reaction (PCR; Canadian patent 1,237,685), and the synthesis of DNA fragments using phosphoramidite chemistry (Matteucci and Caruthers, 1981, J. Am. Chem. Soc.
103:
3185-3191). The application of the reporter gene involves the insertion of the gene into a suitable vector to form double-stranded, circular DNA molecules that are delivered to the eukaryote cells. General methods for the preparation and modification of recombinant DNA molecules have been described by Cohen et al. [U.S. patent No.
4,237,224], Collins et al. [U.S. patent No. 4,304,863], Sambrook et al. (1989, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory), and Mullis and Faloona (1987, Methods Enzymol. 155: 335-350) .
1044-1051). However, none of these intracellular enzymes is suitable as a secreted reporter activity. For instance, the luciferase reporter gene is used widely due mainly to the sensitivity of the detection system and the lack of background activity in most biological systems. However, the luciferase enzyme is relatively unstable under any conditions, and is completely and irreversibly inactivated by the process of secretion.
Secretable human placental alkaline phosphatase (SEAP; a truncated form of membrane-bound alkaline phosphatase; Kam et al., 1985, Proc. Natl. Acad. Sci.
USA
~. .
82: 8715-8719) is a reporter gene that is distributed by Clontech as a secreted reporte:r gene/enzyme system (see European Patent application 0 327 960). However, many biological systems display endogenous (background) alkaline phosphatase (AP) activity.
For example, the high level of alkaline phosphatase activity in cows' milk precludes the use of SEAP as a reporter activity for experiments aimed at using the bovine udder as a "bioreactor" in the production of bioengineered milk. A second commercially-available, secreted reporter gene is human growth hormone (hGH, Boehringer Mannheim; Selden et al., 1986, Mol. Cell. Biol. 6: 31.73-3179 ). However, since hGH:
has no easily measurable biological activity, the detection system for this secreted product uses a laborious, indirect antibody-based ELISA method.
* trademark Thus there is a need for an improved secreted reporter gene that overcomes the problems of the prior art systems. Specifically there is a need for a secreted reporter gene encoding a biological activity that, unlike hGH, is safe and easily measured, but that does not have the background activity problems associated with SEAP.
The chicken a-amylase reporter gene encodes a biological activity that is easily measured by a variety of liquid or semi-solid phase assay systems. The use of the preferred, native gel assay system eliminates any potential interference by endogenous amylase activities produced by the transformed, mammalian target cells. In addition, the advantage of using a secreted reporter activity is that it is not necessary to harvest cells from the culture dish, or to biopsy a transformed tissue (e.g. udder epithelium), nor is it necessary to lyse cells prior to assay. Instead, the cell culture supernatant or biological fluid (e.g. milk) is recovered from the transformed culture or animal and assayed for reporter activity. This feature has several advantages such as the ability to conduct multiple measurements over time on a single transformed population of cells (time course measurements).
The construction of the reporter gene involved the use of standard recombinant DNA methods, including the use of restriction enzymes to fuse DNA molecules witli coherent ends, the amplification of DNA fragments using the polymerase chain reaction (PCR; Canadian patent 1,237,685), and the synthesis of DNA fragments using phosphoramidite chemistry (Matteucci and Caruthers, 1981, J. Am. Chem. Soc.
103:
3185-3191). The application of the reporter gene involves the insertion of the gene into a suitable vector to form double-stranded, circular DNA molecules that are delivered to the eukaryote cells. General methods for the preparation and modification of recombinant DNA molecules have been described by Cohen et al. [U.S. patent No.
4,237,224], Collins et al. [U.S. patent No. 4,304,863], Sambrook et al. (1989, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory), and Mullis and Faloona (1987, Methods Enzymol. 155: 335-350) .
Once assembled, the vector carrying the reporter gene is delivered to the target cells by one of a number of commonly used methods, e.g. viral vectors, calcium phosphate co-precipitation, or liposome-mediated delivery. The DNA plasmid vector used to carry the reporter gene can provide for either transient or stable maintenance of the reporter gene in the target, host cells depending upon the type of vector employed.
The production and secretion of proteins, including reporter enzymes, by eukaryotic cells is a multi-stage process. Briefly, the process begins with the transcription of the gene encoding the secreted product, followed by the processing of the primary transcript into mRNA, and the transport of the mature messenger into the cytoplasm. The mRNA is translated into the preprotein and simultaneously translocated into the first compartment of the secretory pathway by ribosomes associated with the endoplasmic reticulum. Targeting of the preprotein to the secretion pathway is mediated by a secretion signal peptide. Within the golgi apparatus and the secretory vesicles, the primary translation product undergoes sorting and maturation to produce the fmal bioactive protein which is released into the extracellular medium.
The present invention includes a secretion signal sequence that effectively targets the reporter peptide to the secretory machinery of mammalian producer cells.
Any processing signal encrypted within the primary sequence of a heterologous protein which is incompatible with the host cell machinery can lead to a slowdown of the passage of the protein through the secretory pathway, and result in a decrease, from mild to severe, in the efficiency of the production of the secreted product.
The present invention provides for a novel reporter activity based on the chicken a-amylase gene which is compatible with and can be secreted efficiently by mammalian cells.
There are a number of prior reports on the expression and secretion of a-amylase in microbial systems. In addition, Japanese Abstract 63263086 described the use of a-amylase as an indicator gene in a "promoter trap" construct for use in both prokaryote and eukaryote systems. The present system however is preferred, in that this system is designed to measure gene expression and DNA delivery specifically in mammalian cells.
Although a-amylases are available from a number of sources, it has been found according to the present invention that the chicken a-amylase enzyme is preferred.
The chicken a-amylase enzyme is compatible with the mammalian cellular translation and secretion machinery. Therefore, the mammalian cells are able to produce and secrete the chicken enzyme efficiently in an authentic, bioactive form.
Furthermore, the chicken a-amylase has the same physio-chemical requirements for optimal activity and stability, for example, pH optimum, calcium activation, etc., as mannnalian amylases. Therefore the chicken a-amylase is compatible with both the intracellular and extracellular environments of mammalian cells and tissues. This feature is essential for its use as a reporter system. In addition the electrophoretic mobility of the mature, active chicken a-amylase enzyme on native polyacrylamide gels is different from that of mammalian amylases. Therefore if any amylase enzyme is secreted from mammalian cells, the chicken a-amylase reporter activity can easily be differentiated from the background activity. This feature is particularly important when measuring reporter activities in biological fluids, such as cows' milk, which contain high levels of background activity for other reporter systems, e.g. the SEAP reporter system.
In one aspect of the present invention the reporter gene system includes a promoter. When a promoter is included in the system the promoter-containing reporter gene can be used to monitor the efficiency of delivery of foreign DNA
to target cells.
The amount of secreted reporter activity measured in the extracellular medium of a population of cells, transformed with a multicopy plasmid carrying a reporter gene which is driven by a strong, systemic promoter, is determined by two variables: 1) the number of cells that have received the reporter construct and; 2) the average number of copies of the DNA construct that each transformed cell received. Thus, under these conditions, the secreted reporter activity provides a measure of the efficiency of the DNA delivery system, i.e. how many cells in the population were transformed with foreign DNA and how many copies, on average, of the transforming vector were incorporated into each transformed cell.
SUNIlVIARY OF THE INVENTION
Thus according to the present invention there is provided a secreted reporter gene system for mammalian cells. More specifically the present invention relates to a secreted reporter gene system based on chicken a-amylase.
The reporter gene system of the present invention consists of the following components: 1) a signal peptide coding region; 2) a sequence encoding the chicken amylase mature protein; and 3) a transcription termination region.
In one embodiment of the present invention, the reporter gene system comprises a DNA sequence based on the chicken (Gallus gallus) a-amylase gene.
In a further embodiment of the present invention, the signal sequence is based on the Drosophila melanogaster a-amylase secretion signal.
In yet a further embodiment of the present invention the transcription termination region is based on the Drosophila melanogaster a-amylase gene termination region.
In a further aspect of the present invention the reporter gene system also includes a transcription promoter region.
The present invention is also directed to a vector comprising the reporter gene system.
The production and secretion of proteins, including reporter enzymes, by eukaryotic cells is a multi-stage process. Briefly, the process begins with the transcription of the gene encoding the secreted product, followed by the processing of the primary transcript into mRNA, and the transport of the mature messenger into the cytoplasm. The mRNA is translated into the preprotein and simultaneously translocated into the first compartment of the secretory pathway by ribosomes associated with the endoplasmic reticulum. Targeting of the preprotein to the secretion pathway is mediated by a secretion signal peptide. Within the golgi apparatus and the secretory vesicles, the primary translation product undergoes sorting and maturation to produce the fmal bioactive protein which is released into the extracellular medium.
The present invention includes a secretion signal sequence that effectively targets the reporter peptide to the secretory machinery of mammalian producer cells.
Any processing signal encrypted within the primary sequence of a heterologous protein which is incompatible with the host cell machinery can lead to a slowdown of the passage of the protein through the secretory pathway, and result in a decrease, from mild to severe, in the efficiency of the production of the secreted product.
The present invention provides for a novel reporter activity based on the chicken a-amylase gene which is compatible with and can be secreted efficiently by mammalian cells.
There are a number of prior reports on the expression and secretion of a-amylase in microbial systems. In addition, Japanese Abstract 63263086 described the use of a-amylase as an indicator gene in a "promoter trap" construct for use in both prokaryote and eukaryote systems. The present system however is preferred, in that this system is designed to measure gene expression and DNA delivery specifically in mammalian cells.
Although a-amylases are available from a number of sources, it has been found according to the present invention that the chicken a-amylase enzyme is preferred.
The chicken a-amylase enzyme is compatible with the mammalian cellular translation and secretion machinery. Therefore, the mammalian cells are able to produce and secrete the chicken enzyme efficiently in an authentic, bioactive form.
Furthermore, the chicken a-amylase has the same physio-chemical requirements for optimal activity and stability, for example, pH optimum, calcium activation, etc., as mannnalian amylases. Therefore the chicken a-amylase is compatible with both the intracellular and extracellular environments of mammalian cells and tissues. This feature is essential for its use as a reporter system. In addition the electrophoretic mobility of the mature, active chicken a-amylase enzyme on native polyacrylamide gels is different from that of mammalian amylases. Therefore if any amylase enzyme is secreted from mammalian cells, the chicken a-amylase reporter activity can easily be differentiated from the background activity. This feature is particularly important when measuring reporter activities in biological fluids, such as cows' milk, which contain high levels of background activity for other reporter systems, e.g. the SEAP reporter system.
In one aspect of the present invention the reporter gene system includes a promoter. When a promoter is included in the system the promoter-containing reporter gene can be used to monitor the efficiency of delivery of foreign DNA
to target cells.
The amount of secreted reporter activity measured in the extracellular medium of a population of cells, transformed with a multicopy plasmid carrying a reporter gene which is driven by a strong, systemic promoter, is determined by two variables: 1) the number of cells that have received the reporter construct and; 2) the average number of copies of the DNA construct that each transformed cell received. Thus, under these conditions, the secreted reporter activity provides a measure of the efficiency of the DNA delivery system, i.e. how many cells in the population were transformed with foreign DNA and how many copies, on average, of the transforming vector were incorporated into each transformed cell.
SUNIlVIARY OF THE INVENTION
Thus according to the present invention there is provided a secreted reporter gene system for mammalian cells. More specifically the present invention relates to a secreted reporter gene system based on chicken a-amylase.
The reporter gene system of the present invention consists of the following components: 1) a signal peptide coding region; 2) a sequence encoding the chicken amylase mature protein; and 3) a transcription termination region.
In one embodiment of the present invention, the reporter gene system comprises a DNA sequence based on the chicken (Gallus gallus) a-amylase gene.
In a further embodiment of the present invention, the signal sequence is based on the Drosophila melanogaster a-amylase secretion signal.
In yet a further embodiment of the present invention the transcription termination region is based on the Drosophila melanogaster a-amylase gene termination region.
In a further aspect of the present invention the reporter gene system also includes a transcription promoter region.
The present invention is also directed to a vector comprising the reporter gene system.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIGURE 1 shows the sequence of the chicken a-amylase enzyme (SEQID No.: 1).
FIGURE 2 shows an example of the reporter gene construct which does not contain a promoter region. The components of the reporter gene are shown as shaded rectangles. The transcription promoter 'pro', a-amylase mature peptide coding sequence 'amylase', and transcription termination region 'ter' are indicated.
The solid black rectangle between the promoter and the amylase coding region represents the secretion signal peptide. Restriction enzyme recognition sites are as follows: As = AscI ; Hi = HindIII; Nc = Nco1; Nh = Nhel; No = Not1.
The 8-cutter sites Not1 and Ascl were built into the upstream and downstream ends, respectively, of the reporter gene in order to facilitate its insertion into the reporter construct.
FIGURE 3 shows an example of the reporter gene construct which contains a promoter region. The symbols are as described for Figure 2.
FIGURE 4 shows the sequence of the transcription termination region (SEQID
No.:
2).
FIGURE 5 shows the electrophoretic mobilities of a-amylases from 3 different sources.
FIGURE 6 shows the production of the reporter activity by manunalian cells in culture.
FIGURE 7 shows the detection of amylase activity in cows' milk.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIGURE 1 shows the sequence of the chicken a-amylase enzyme (SEQID No.: 1).
FIGURE 2 shows an example of the reporter gene construct which does not contain a promoter region. The components of the reporter gene are shown as shaded rectangles. The transcription promoter 'pro', a-amylase mature peptide coding sequence 'amylase', and transcription termination region 'ter' are indicated.
The solid black rectangle between the promoter and the amylase coding region represents the secretion signal peptide. Restriction enzyme recognition sites are as follows: As = AscI ; Hi = HindIII; Nc = Nco1; Nh = Nhel; No = Not1.
The 8-cutter sites Not1 and Ascl were built into the upstream and downstream ends, respectively, of the reporter gene in order to facilitate its insertion into the reporter construct.
FIGURE 3 shows an example of the reporter gene construct which contains a promoter region. The symbols are as described for Figure 2.
FIGURE 4 shows the sequence of the transcription termination region (SEQID
No.:
2).
FIGURE 5 shows the electrophoretic mobilities of a-amylases from 3 different sources.
FIGURE 6 shows the production of the reporter activity by manunalian cells in culture.
FIGURE 7 shows the detection of amylase activity in cows' milk.
DESCRIPTION OF PREFERRED EMBODIMENT
Alpha-amylases are enzymes that require secretion to achieve biological activity and are very stable once secreted. The enzyme activity is easily measured by a variety of assays, including the simple, inexpensive and sensitive native gel-electrophoresis assay, which has an additional advantage of discriminating between different electrophoretic variants of the enzyme. Thus, although endogenous activity is usually not a problem, if the biological system under investigation produces a background amylase activity, one can easily differentiate between the endogenous activity and the activity encoded by the reporter gene using the gel separation assay.
Selection of cell colonies producing amylase activity can also be accomplished using a simple clearing zone assay that is performed by diffusion in starch-containing medium.
Included within the scope of the present invention are modified DNA sequences encoding a functionally active a-amylase gene. Possible modifications include but are not limited to: 1) nucleotide substitutions that eliminate restriction enzyme (RE) sites in the naturally-occurring sequence, but do not alter the amino acid sequences, and the use of these RE sites in a multiple-cloning-region for the introduction of DNA
components, e.g. the transcription promoter; 2) the enhancement of the specific activity of the reporter peptide by amino acid substitutions; 3) increasing the temperature stability of the reporter enzyme by amino acid substitutions. Also included is the potential use of amylase inhibitors, isolated from plant sources, to specifically inhibit any endogenous, mammalian amylases that might be present in the extracellular medium of the transformed cells.
The reporter gene system of the present invention also includes a DNA
sequence encoding a signal peptide coding region which is based on the Drosophila melanogaster a-amylase pre-protein. This signal peptide works effectively in combination with the chicken a-amylase enzyme and the mammalian cellular secretion machinery. The DNA sequence encoding the signal peptide was modified to include useful restriction enzyme sites for the introduction of promoter sequences to drive the reporter gene. The present invention also encompasses any modifications of the signal peptide region that may increase the efficiency of the secretion process beyond the current level.
The reporter gene system of the present invention also includes a transcription termination region. In one embodiment of the present invention the transcription terminator region is derived from the Drosophila melanogaster amylase gene.
This portion of the reporter gene serves the following functions: (1) to provide a 3' non-coding region for the transcript; (2) to provide a transcription termination signal for the RNA polymerase complex; and (3) to provide a poly-adenylation motif to the primary transcript. Included within the scope of the present invention is the addition of any functional DNA elements, such as transcription enhancer elements, introns, etc., to the transcription terminator region in order to enhance the overall level of expression of the reporter gene.
In one embodiment of the present invention, the reporter gene system also includes a DNA sequence encoding a transcription promoter region. When the reporter gene system of the present invention includes a promoter, the primary use of this secreted reporter gene system is to monitor the efficiency of the introduction of foreign DNA into mammalian cells in vivo, in vitro and ex vivo. This application facilitates experiments designed to optimize the conditions, methods and vehicles employed for the introduction of foreign DNA into mammalian cells. There are a number of high expression level, commercially available promoters which are suitable for this application.
One aspect of the invention would be the use of a tissue-specific promoter to drive the expression of the reporter gene. This configuration would enable the researcher to distinguish between expression in target and non-target cell populations.
Furthermore, it would allow researchers to optimize their delivery systems for targeting the foreign DNA to cell types that have the greatest capacity for producing and secreting foreign peptides. This aspect is especially important for in vivo gene transfer. Specific applications would include gene transfer to udder epithelial cells in mammals for the production of pharmaceutical agents into the milk (the somatic bioreactor concept), and DNA transfer for gene therapy in humans.
While this invention is described in detail with particular reference to preferred embodiments thereof, said embodiments are offered to illustrate but do not limit the invention.
EXAMPLES
EXAMPLE 1: Chicken a-amylase cDNA
In order to isolate a chicken genoniic a-amylase clone, a commercial chicken library in the vector a. FIX II(Stratagene) was screened with a murine amylase cDNA
probe (1.2 kbp PstI fragment of pMPa2l, Hagenbuechle et al., 1980, Cell 21:
187). Hybridization was carried out under conditions of reduced stringency at in a standard buffer system containing 50% formamide (Benkel and Gavora, 1993, Animal Genetics 24: 409-413). Following hybridization, the filters were washed twice for 15 min each in 2x SSC, 0.1 % SDS at 42 C, followed by a single wash in 0.5x SSC, 0.1 % SDS at 50 C for 30 min. Autoradiograms were exposed at -70 C with intensifying screens.
Eighteen positive signals were detected following the first round of library screening. Ten plaques were chosen at random for second screening, and 4 of these isolates were still positive after a third round of screening. One clone (XA-1) was chosen for large-scale DNA isolation and fragment subcloning in preparation for sequence analysis.
A series of overlapping subclones spanning the amylase genomic region was constructed by inserting restriction fragments of the primary isolate .lA-1 into the vector pUC18. Double-stranded DNA was sequenced by the gene-walking method using synthetic oligonucleotide primers. Primers were synthesized on an Applied Biosystems 392 synthesizer and deblocked and desalted before use. Sequencing reactions were performed using the dye terminator cycle sequencing kit as described in the instructions supplied by the manufacturer (Applied Biosystems'Inc.).
The extension products were analyzed on an Applied Biosystems 373A~ automated sequencer, and sequence assembly was performed using MicroGenie software by Beckman.
The chicken amylase coding region (SEQID No.: 1) was prepared using reverse transcription-polymerase chain reaction technology (RT-PCR). Oligonucleotide primers for the PCR step were designed based on the chicken genomic amylase derived as described above. Approximately 2 g of total RNA from chicken pancreas was used as substrate in a reverse transcription (RT) reaction using the Perkin Elmer RT-PCR kit components according to the instructions supplied by the manufacturer.
The RT-reaction was primed with oligo-dT. The oligonu.cleotide primers used for the PCR
stage of the RT-PCR reaction were as follows :(i) Chamy-Nhe (5' -ATQCJ-AGCTCAGTACAATCCCAACACTCAGGCT-3' ; SEQ ID No.:3) which spans the position in the coding region corresponding to the N-terminus of the mature enzyme; and (ii) Chamy-Hin (5'-CGAAGC'~ATAACTTGGCATCA
ACGTGAATTG-3'; SEQ ID No.: 4) which spans the stop codon of the amylase coding region. The primers were designed to amplify the region in the gene encoding the mature a-amylase peptide. In addition, Chamy-Nhe converts the environment of the signal peptidase cleavage site into an Nhel site, while Chamy-Hin adds a HindIII
site immediately downstream of the stop codon (enzyme recognition sites are underlined). The modifications introduced by the PCR primers allow the amplified cDNA to be inserted into the reporter construct using cohesive overhang ligation.
PCR amplification of the chicken amylase cDNA was performed using the LA-PCR kit as described in the manufacturer's product bulletin (TaKaRa). The amplified fragment was sequenced using PCR-based chain termination technology, prior to insertion into the reporter construct - this sequence is shown in Figure 1.
trademark I e -~, ~~= ...-_-.-..,.-~._,._- - - --_ ,.._ ._..~.w . , . ...,,...,,.,...,..
...::. ,. . ,...,.:.... .. . .. ,..... ., .,......,.,..A ..>. ..
..,...,_.__...._... . .._....._.__ _ .. .__.........-.._._.. ~ _. . ......_...
. ..__...._._. ... __ ..
Alpha-amylases are enzymes that require secretion to achieve biological activity and are very stable once secreted. The enzyme activity is easily measured by a variety of assays, including the simple, inexpensive and sensitive native gel-electrophoresis assay, which has an additional advantage of discriminating between different electrophoretic variants of the enzyme. Thus, although endogenous activity is usually not a problem, if the biological system under investigation produces a background amylase activity, one can easily differentiate between the endogenous activity and the activity encoded by the reporter gene using the gel separation assay.
Selection of cell colonies producing amylase activity can also be accomplished using a simple clearing zone assay that is performed by diffusion in starch-containing medium.
Included within the scope of the present invention are modified DNA sequences encoding a functionally active a-amylase gene. Possible modifications include but are not limited to: 1) nucleotide substitutions that eliminate restriction enzyme (RE) sites in the naturally-occurring sequence, but do not alter the amino acid sequences, and the use of these RE sites in a multiple-cloning-region for the introduction of DNA
components, e.g. the transcription promoter; 2) the enhancement of the specific activity of the reporter peptide by amino acid substitutions; 3) increasing the temperature stability of the reporter enzyme by amino acid substitutions. Also included is the potential use of amylase inhibitors, isolated from plant sources, to specifically inhibit any endogenous, mammalian amylases that might be present in the extracellular medium of the transformed cells.
The reporter gene system of the present invention also includes a DNA
sequence encoding a signal peptide coding region which is based on the Drosophila melanogaster a-amylase pre-protein. This signal peptide works effectively in combination with the chicken a-amylase enzyme and the mammalian cellular secretion machinery. The DNA sequence encoding the signal peptide was modified to include useful restriction enzyme sites for the introduction of promoter sequences to drive the reporter gene. The present invention also encompasses any modifications of the signal peptide region that may increase the efficiency of the secretion process beyond the current level.
The reporter gene system of the present invention also includes a transcription termination region. In one embodiment of the present invention the transcription terminator region is derived from the Drosophila melanogaster amylase gene.
This portion of the reporter gene serves the following functions: (1) to provide a 3' non-coding region for the transcript; (2) to provide a transcription termination signal for the RNA polymerase complex; and (3) to provide a poly-adenylation motif to the primary transcript. Included within the scope of the present invention is the addition of any functional DNA elements, such as transcription enhancer elements, introns, etc., to the transcription terminator region in order to enhance the overall level of expression of the reporter gene.
In one embodiment of the present invention, the reporter gene system also includes a DNA sequence encoding a transcription promoter region. When the reporter gene system of the present invention includes a promoter, the primary use of this secreted reporter gene system is to monitor the efficiency of the introduction of foreign DNA into mammalian cells in vivo, in vitro and ex vivo. This application facilitates experiments designed to optimize the conditions, methods and vehicles employed for the introduction of foreign DNA into mammalian cells. There are a number of high expression level, commercially available promoters which are suitable for this application.
One aspect of the invention would be the use of a tissue-specific promoter to drive the expression of the reporter gene. This configuration would enable the researcher to distinguish between expression in target and non-target cell populations.
Furthermore, it would allow researchers to optimize their delivery systems for targeting the foreign DNA to cell types that have the greatest capacity for producing and secreting foreign peptides. This aspect is especially important for in vivo gene transfer. Specific applications would include gene transfer to udder epithelial cells in mammals for the production of pharmaceutical agents into the milk (the somatic bioreactor concept), and DNA transfer for gene therapy in humans.
While this invention is described in detail with particular reference to preferred embodiments thereof, said embodiments are offered to illustrate but do not limit the invention.
EXAMPLES
EXAMPLE 1: Chicken a-amylase cDNA
In order to isolate a chicken genoniic a-amylase clone, a commercial chicken library in the vector a. FIX II(Stratagene) was screened with a murine amylase cDNA
probe (1.2 kbp PstI fragment of pMPa2l, Hagenbuechle et al., 1980, Cell 21:
187). Hybridization was carried out under conditions of reduced stringency at in a standard buffer system containing 50% formamide (Benkel and Gavora, 1993, Animal Genetics 24: 409-413). Following hybridization, the filters were washed twice for 15 min each in 2x SSC, 0.1 % SDS at 42 C, followed by a single wash in 0.5x SSC, 0.1 % SDS at 50 C for 30 min. Autoradiograms were exposed at -70 C with intensifying screens.
Eighteen positive signals were detected following the first round of library screening. Ten plaques were chosen at random for second screening, and 4 of these isolates were still positive after a third round of screening. One clone (XA-1) was chosen for large-scale DNA isolation and fragment subcloning in preparation for sequence analysis.
A series of overlapping subclones spanning the amylase genomic region was constructed by inserting restriction fragments of the primary isolate .lA-1 into the vector pUC18. Double-stranded DNA was sequenced by the gene-walking method using synthetic oligonucleotide primers. Primers were synthesized on an Applied Biosystems 392 synthesizer and deblocked and desalted before use. Sequencing reactions were performed using the dye terminator cycle sequencing kit as described in the instructions supplied by the manufacturer (Applied Biosystems'Inc.).
The extension products were analyzed on an Applied Biosystems 373A~ automated sequencer, and sequence assembly was performed using MicroGenie software by Beckman.
The chicken amylase coding region (SEQID No.: 1) was prepared using reverse transcription-polymerase chain reaction technology (RT-PCR). Oligonucleotide primers for the PCR step were designed based on the chicken genomic amylase derived as described above. Approximately 2 g of total RNA from chicken pancreas was used as substrate in a reverse transcription (RT) reaction using the Perkin Elmer RT-PCR kit components according to the instructions supplied by the manufacturer.
The RT-reaction was primed with oligo-dT. The oligonu.cleotide primers used for the PCR
stage of the RT-PCR reaction were as follows :(i) Chamy-Nhe (5' -ATQCJ-AGCTCAGTACAATCCCAACACTCAGGCT-3' ; SEQ ID No.:3) which spans the position in the coding region corresponding to the N-terminus of the mature enzyme; and (ii) Chamy-Hin (5'-CGAAGC'~ATAACTTGGCATCA
ACGTGAATTG-3'; SEQ ID No.: 4) which spans the stop codon of the amylase coding region. The primers were designed to amplify the region in the gene encoding the mature a-amylase peptide. In addition, Chamy-Nhe converts the environment of the signal peptidase cleavage site into an Nhel site, while Chamy-Hin adds a HindIII
site immediately downstream of the stop codon (enzyme recognition sites are underlined). The modifications introduced by the PCR primers allow the amplified cDNA to be inserted into the reporter construct using cohesive overhang ligation.
PCR amplification of the chicken amylase cDNA was performed using the LA-PCR kit as described in the manufacturer's product bulletin (TaKaRa). The amplified fragment was sequenced using PCR-based chain termination technology, prior to insertion into the reporter construct - this sequence is shown in Figure 1.
trademark I e -~, ~~= ...-_-.-..,.-~._,._- - - --_ ,.._ ._..~.w . , . ...,,...,,.,...,..
...::. ,. . ,...,.:.... .. . .. ,..... ., .,......,.,..A ..>. ..
..,...,_.__...._... . .._....._.__ _ .. .__.........-.._._.. ~ _. . ......_...
. ..__...._._. ... __ ..
EXAMPLE 2: Signal Peptide Coding Region The signal peptide used in the current reporter gene is modelled on the sequence found in the Amyl gene of the Oregon-R strain of Drosophila melanogaster (Boer and Hickey, 1986, Nuc. Acids Res. 14: 8399-8411). The sequence of the native signal peptide extends from nucleotide 1 to 54 in Figure 2 of that reference. The 5'-end of the sequence encoding the signal peptide was modified to incorporate an NcoI site straddling the translation start codon (ATG) - i.e. the sequence AT ATG T was changed to CC ATG
G. This modification changes the amino acid immediately downstream of the initiator Met from a Phe to a Val. In addition, the 3'-end of the signal sequence was modified to accommodate an NheI site. Here, the original D. melanogaster sequence of --Ala-Asn-Ala-- (GCC AAC GCC) was changed to --Ala-Leu-Ala-- (GCG CTA GCC).
The DNA fragment encoding the signal peptide was constructed in the laboratory on a DNA synthesizer, and assembled into the reporter construct using the restriction enzyme sites built into its ends. The novel signal peptide sequence used in the reporter construct has the following sequence (SEQID No.: 5).
5'-CCATGGTTCTGGCCAAGAGCATAGTGTGCCTCGCCCTCCTGGCGGTGGCGCTAGCT-3' 3'-GGTACCAAGACCGGTTCTCGTATCACACGGAGCGGGAGGACCGCCACCGCGATCGA-5' Ncol Ntiel EXAMPLE 3: Promoter Region In this example the immediate early promoter of the Human cytomegalovirus (CMV; Stinski and Roehr, 1985, J. Virology 55: 431-441) was used to drive the expression of the reporter gene.
Other examples of promoters that have been used successfully in the reporter gene include; a murine retroviral LTR, which is a composite of the MMTV (Mouse Mammary Tumor Virus; Ponta et al. 1985, Proc. Natl. Acad. Sci. 82: 1020-1024) and the MoMSV (Moloney Murine Sarcoma Virus; Lin et al. 1990, Proc. Natl. Acad.
Sci. 87:
G. This modification changes the amino acid immediately downstream of the initiator Met from a Phe to a Val. In addition, the 3'-end of the signal sequence was modified to accommodate an NheI site. Here, the original D. melanogaster sequence of --Ala-Asn-Ala-- (GCC AAC GCC) was changed to --Ala-Leu-Ala-- (GCG CTA GCC).
The DNA fragment encoding the signal peptide was constructed in the laboratory on a DNA synthesizer, and assembled into the reporter construct using the restriction enzyme sites built into its ends. The novel signal peptide sequence used in the reporter construct has the following sequence (SEQID No.: 5).
5'-CCATGGTTCTGGCCAAGAGCATAGTGTGCCTCGCCCTCCTGGCGGTGGCGCTAGCT-3' 3'-GGTACCAAGACCGGTTCTCGTATCACACGGAGCGGGAGGACCGCCACCGCGATCGA-5' Ncol Ntiel EXAMPLE 3: Promoter Region In this example the immediate early promoter of the Human cytomegalovirus (CMV; Stinski and Roehr, 1985, J. Virology 55: 431-441) was used to drive the expression of the reporter gene.
Other examples of promoters that have been used successfully in the reporter gene include; a murine retroviral LTR, which is a composite of the MMTV (Mouse Mammary Tumor Virus; Ponta et al. 1985, Proc. Natl. Acad. Sci. 82: 1020-1024) and the MoMSV (Moloney Murine Sarcoma Virus; Lin et al. 1990, Proc. Natl. Acad.
Sci. 87:
36-40) LTRs, and the rat beta-actin promoter (Nudel et al. 1983, Nucl. Acids Res., 11, 1759-1771).
DNA fragments to be used as promoters were amplified by PCR using oligonucleotide primers that incorporated useful restriction enzyme sites at the upstream (NotI) and downstream (Ncol) ends of the amplified promoter sequences.
Restricted promoters fragments were inserted into the reporter gene construct by cohesive end ligation.
EXAMPLE 4: Transcription Termination Region The fmal component of the reporter gene is the transcription terminator. In this example 567 bp of the Drosophila melanogaster Oregon-R amylase gene tennination region was used. The sequence of this section (SEQID No: 2) is shown in Figure 4. The DNA fragment was amplified by PCR using primers that incorporated useful restriction enzyme sites. A HindIII site was added just upstream of the TAA (stop codon) of the amylase coding region. This TAA is out of frame with the chicken amylase coding region, which brings in its own stop codon. The downstream end of the terminator is modified into an Ascl site.
EXAMPLE 5: The Chicken a-amylase reporter gene system Delivery of Transforming DNA
A variety of methods is available for the delivery of the reporter gene to the target host cells including: 1) liposome-mediated fusion; 2) micro-injection;
3) particle bombardment; 4) viral vectors; and 5) calcium phosphate co-precipitation.
For the purpose of this non-limiting illustrative example, we have inserted the reporter gene linked to the CMV promoter into the plasmid pIBI25 (International Biotechnologies Incorporated) to produce a transient expression construct. In addition, we have delivered the reporter construct to the cells using the calcium phosphate co-precipitation method. Other methods of DNA delivery are applicable, as is the use of vectors that promote the stable maintenance of the reporter gene within the transformed cells, e.g. retroviral vectors.
Harvesting of the Reporter Protein Cells were grown under standard mammalian cell culture conditions. The media used to culture the cell lines were as follows: (i) for MA104 and Hela, Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal Bovine Serum; (ii) for CHO, DMEM with 10% Newborn Calf Serum; (iii) for PA317 and PG13, DMEM
with iron-enriched 10% Calf Serum. Cells were plated at a density of 5x105 cells per 10 cm dish. Calcium phosphate transfection was carried out overnight.
Following transfection, cells were incubated overnight in cell line-specific media as described above. On day 4, the culture medium was removed and replaced with Serum-free DMEM for all cell lines. On day 5, samples of medium were collected, debris was removed from the samples by centrifugation at 500xg, and the supernatants prepared for amylase gel analysis.
For the gel assay, 1 mL of cell supernatant was adjusted to 40% (vol/vol) of ethanol and the mixture incubated on ice for 1 hour. The solution was centrifuged at 15,000xg for 20 minutes and the supernatant dried under vacum to a fmal volume of 1 mL to remove the ethanol. The resulting solution was concentrated to 100 uL
using centricon-30 filtration columns (Amicon) and the samples analyzed for amylase activity.
Detection of Reporter Activity Amylase activity can be detected and measured by a variety of simple, safe procedures. These can be divided into the tube or liquid assays and the assays that involve diffasion in a semi-solid medium or electrophoretic separation.
The prefererd protocol is electrophoretic separation of the proteins in the sample on a native (non-denaturing) polyacrylaniide gel. This protocol is described in Benkel and Hickey (1986, Genetics 114: 137-144). Briefly, a low percentage acrylamide gel allows proteins to be separated on the basis of the overall charge of the molecules. Thus, even though chicken and manunalian amylases are very similar in molecular weight, they display very different migration patterns in the native gel assay (see Figure 5).
Following electrophoretic separation, the gel is incubated in a buffer solution containing partially-hydrolyzed starch. The starch granules coat the gel and penetrate the gel surface. Staining of the starch-coated gel with iodine results in a gel that shows clear amylase bands on a dark blue background.
The main advantage of the gel assay is that the background activity measured for the recipient cells is essentially zero. The gel assay is highly sensitive, and can easily be converted into a quantitative format by the incorporation of serial dilutions of an activity standard (see Benkel and Hickey, 1986, Genetics 114: 943-954).
In addition, there is a dye-linked starch-based substrate available that real-time activity visualization.
We have tested chicken amylase in cell culture medium and in milk, and have found the activity to be stable for days at room temperature in bioactive format. In addition, freezing and thawing does not affect the activity of the enzyme.
Results of In Vitro Tests Figure 6 shows a variety of mammalian cell lines transformed with the reporter construct containing the CMV promoter delivered using a transient-expression vector.
All cell lines tested including; CHO (hamster), ET-2 (cow), Hela (human), (monkey), PA317 and PG13 (mouse) showed production and secretion of chicken amylase according to the native gel assay. Different cell lines appear to secrete chicken amylase at different efficiencies. This may reflect the ability of the cells to take up foreign DNA, or it may be a measure of the inherent capacity of the different cell types to secrete proteins.
Secreted Reporter Enzymes in Cows' Milk One of the primary applications for secreted reporter genes is in the optimization of DNA delivery and transformation in vivo of mammalian cells that secrete proteins in to biological fluids. This optimization process is crucial to the development of successful approaches for cell transformation in gene therapy.
We are currently exploring the use of somatic transgenesis of bovine epithelial tissue in the development of 'bioreactor' cows for the production of pharmaceutical agents into milk. In order to compare the suitabilities of SEAP and a-amylase as reporter gene/enzyme systems for the transformation of cow udder cells, we performed a set of reconstitution experiments in which SEAP and amylase activities were measured in fresh cows' milk with and without the addition of preparates containing easily measurable quantities of SEAP and amylase enzymes (spiked samples).
Fresh cows' milk was centrifuged at 8,000 xg for 10 minutes at room temperature and the aqueous fraction recovered and used for SEAP and amylase activity detenninations. For the SEAP reconstitution experiment, SEAP activity was measured for the following treatments: (i) untreated milk aqueous phase ("Endogenous" in Table 1); (ii) milk aqueous phase heated to 65 C for 30 minutes ("Heated"); (iii) milk aqueous phase with SEAP spike ("Spiked"); and (iv) milk aqueous phase with SEAP spike heated to 65 C for 30 minutes ("Spiked/heated").
The results demonstrated that there is a high background of endogenous, soluble alkaline phosphatase activity in cows' milk. This activity is indistinguishable from SEAP. Heating the samples to 65 C does not decrease the endogenous SEAP
activity level; instead it appears that a portion of the endogenous alkaline phosphatase activity is present in an inactive, protein-complexed state in milk, and that heating tends to promote the release of some of this protein-bound endogenous SEAP activity.
This results in a further increase in the background SEAP levels measured in the unspiked milk. The ability of milk proteins to inactivate SEAP is also evident from the dramatic decrease in SEAP signal measured in the spiked samples, where only about 1/4 of the SEAP activity added in the spike is detectable following the addition of the milk.
Heating the spiked samples to 65 C raises the levels somewhat on average, but this increase in activity is at least partially due to the release of endogenous alkaline phosphatase enzyme.
Table 1: Measurement of SEAP activity in cows, milk.
Sample Endogenous Heated Spiked Spiked/heated Cow 1 52 60 282 307 Cow 2 59 62 198 190 Legend: Cows' milk was processed as described in the text. SEAP activities are given in 1000's of luminometer units. "Endogenous" refers to the endogenous background activity present in the aqueous phase of the milk; "Heated" refers to the background activity in the milk following a 30 minute incubation at 65 C; "Spiked" refers to the unheated milk following the addition of the SEAP spike; "Spiked/heated"
represents the spiked sample following a 30 minute incubation at 65 C. The SEAP spike consisted of 1,075 (x1000) units.
Increasing the level of the alkaline phosphatase-inhibitor homoarginine in the samples had no effect on the SEAP activity measurements. On the other hand, acid curdling of the milk to remove caseins, a standard method used to fractionate milk, resulted in the loss of the bulk of the SEAP spike.
Figure 7 shows milk samples with and without the addition of chicken a-amylase preparations. For whole milk, the gel assay shows a light smear throughout the entire lane (see lane 1). This is due to the effect of the abundant milk casein proteins. However, the band of activity corresponding to the chicken amylase reporter 'spike' is clearly visible even in whole milk (lane 2). Our standard protocol for the amylase reconstitution experiments included an acid curdling step to remove caseins from the whole milk. This treatment was incompatible with the SEAP reporter system because it almost entirely depleted the milk aqueous phase of alkaline phosphatase activity (see above).
Following neutralization and centrifugation of the milk containing the chicken a-amylase spike, the samples were concentrated and analyzed by the standard gel assay. The removal of the caseins completely eliminated any endogenous background activity from the milk samples, and left the chicken amylase band clearly visible in the spiked samples (lanes 7&8). Thus, chicken a-amylase is superior to SEAP as a secreted reporter enzyme in biological fluids such as cow's milk.
15 The present invention has been described with regard to preferred embodiments.
However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described in the following claims.
- .._.. ... r.,~~, ~,. <..r, . ~,~,.,. ,...~ .
DNA fragments to be used as promoters were amplified by PCR using oligonucleotide primers that incorporated useful restriction enzyme sites at the upstream (NotI) and downstream (Ncol) ends of the amplified promoter sequences.
Restricted promoters fragments were inserted into the reporter gene construct by cohesive end ligation.
EXAMPLE 4: Transcription Termination Region The fmal component of the reporter gene is the transcription terminator. In this example 567 bp of the Drosophila melanogaster Oregon-R amylase gene tennination region was used. The sequence of this section (SEQID No: 2) is shown in Figure 4. The DNA fragment was amplified by PCR using primers that incorporated useful restriction enzyme sites. A HindIII site was added just upstream of the TAA (stop codon) of the amylase coding region. This TAA is out of frame with the chicken amylase coding region, which brings in its own stop codon. The downstream end of the terminator is modified into an Ascl site.
EXAMPLE 5: The Chicken a-amylase reporter gene system Delivery of Transforming DNA
A variety of methods is available for the delivery of the reporter gene to the target host cells including: 1) liposome-mediated fusion; 2) micro-injection;
3) particle bombardment; 4) viral vectors; and 5) calcium phosphate co-precipitation.
For the purpose of this non-limiting illustrative example, we have inserted the reporter gene linked to the CMV promoter into the plasmid pIBI25 (International Biotechnologies Incorporated) to produce a transient expression construct. In addition, we have delivered the reporter construct to the cells using the calcium phosphate co-precipitation method. Other methods of DNA delivery are applicable, as is the use of vectors that promote the stable maintenance of the reporter gene within the transformed cells, e.g. retroviral vectors.
Harvesting of the Reporter Protein Cells were grown under standard mammalian cell culture conditions. The media used to culture the cell lines were as follows: (i) for MA104 and Hela, Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal Bovine Serum; (ii) for CHO, DMEM with 10% Newborn Calf Serum; (iii) for PA317 and PG13, DMEM
with iron-enriched 10% Calf Serum. Cells were plated at a density of 5x105 cells per 10 cm dish. Calcium phosphate transfection was carried out overnight.
Following transfection, cells were incubated overnight in cell line-specific media as described above. On day 4, the culture medium was removed and replaced with Serum-free DMEM for all cell lines. On day 5, samples of medium were collected, debris was removed from the samples by centrifugation at 500xg, and the supernatants prepared for amylase gel analysis.
For the gel assay, 1 mL of cell supernatant was adjusted to 40% (vol/vol) of ethanol and the mixture incubated on ice for 1 hour. The solution was centrifuged at 15,000xg for 20 minutes and the supernatant dried under vacum to a fmal volume of 1 mL to remove the ethanol. The resulting solution was concentrated to 100 uL
using centricon-30 filtration columns (Amicon) and the samples analyzed for amylase activity.
Detection of Reporter Activity Amylase activity can be detected and measured by a variety of simple, safe procedures. These can be divided into the tube or liquid assays and the assays that involve diffasion in a semi-solid medium or electrophoretic separation.
The prefererd protocol is electrophoretic separation of the proteins in the sample on a native (non-denaturing) polyacrylaniide gel. This protocol is described in Benkel and Hickey (1986, Genetics 114: 137-144). Briefly, a low percentage acrylamide gel allows proteins to be separated on the basis of the overall charge of the molecules. Thus, even though chicken and manunalian amylases are very similar in molecular weight, they display very different migration patterns in the native gel assay (see Figure 5).
Following electrophoretic separation, the gel is incubated in a buffer solution containing partially-hydrolyzed starch. The starch granules coat the gel and penetrate the gel surface. Staining of the starch-coated gel with iodine results in a gel that shows clear amylase bands on a dark blue background.
The main advantage of the gel assay is that the background activity measured for the recipient cells is essentially zero. The gel assay is highly sensitive, and can easily be converted into a quantitative format by the incorporation of serial dilutions of an activity standard (see Benkel and Hickey, 1986, Genetics 114: 943-954).
In addition, there is a dye-linked starch-based substrate available that real-time activity visualization.
We have tested chicken amylase in cell culture medium and in milk, and have found the activity to be stable for days at room temperature in bioactive format. In addition, freezing and thawing does not affect the activity of the enzyme.
Results of In Vitro Tests Figure 6 shows a variety of mammalian cell lines transformed with the reporter construct containing the CMV promoter delivered using a transient-expression vector.
All cell lines tested including; CHO (hamster), ET-2 (cow), Hela (human), (monkey), PA317 and PG13 (mouse) showed production and secretion of chicken amylase according to the native gel assay. Different cell lines appear to secrete chicken amylase at different efficiencies. This may reflect the ability of the cells to take up foreign DNA, or it may be a measure of the inherent capacity of the different cell types to secrete proteins.
Secreted Reporter Enzymes in Cows' Milk One of the primary applications for secreted reporter genes is in the optimization of DNA delivery and transformation in vivo of mammalian cells that secrete proteins in to biological fluids. This optimization process is crucial to the development of successful approaches for cell transformation in gene therapy.
We are currently exploring the use of somatic transgenesis of bovine epithelial tissue in the development of 'bioreactor' cows for the production of pharmaceutical agents into milk. In order to compare the suitabilities of SEAP and a-amylase as reporter gene/enzyme systems for the transformation of cow udder cells, we performed a set of reconstitution experiments in which SEAP and amylase activities were measured in fresh cows' milk with and without the addition of preparates containing easily measurable quantities of SEAP and amylase enzymes (spiked samples).
Fresh cows' milk was centrifuged at 8,000 xg for 10 minutes at room temperature and the aqueous fraction recovered and used for SEAP and amylase activity detenninations. For the SEAP reconstitution experiment, SEAP activity was measured for the following treatments: (i) untreated milk aqueous phase ("Endogenous" in Table 1); (ii) milk aqueous phase heated to 65 C for 30 minutes ("Heated"); (iii) milk aqueous phase with SEAP spike ("Spiked"); and (iv) milk aqueous phase with SEAP spike heated to 65 C for 30 minutes ("Spiked/heated").
The results demonstrated that there is a high background of endogenous, soluble alkaline phosphatase activity in cows' milk. This activity is indistinguishable from SEAP. Heating the samples to 65 C does not decrease the endogenous SEAP
activity level; instead it appears that a portion of the endogenous alkaline phosphatase activity is present in an inactive, protein-complexed state in milk, and that heating tends to promote the release of some of this protein-bound endogenous SEAP activity.
This results in a further increase in the background SEAP levels measured in the unspiked milk. The ability of milk proteins to inactivate SEAP is also evident from the dramatic decrease in SEAP signal measured in the spiked samples, where only about 1/4 of the SEAP activity added in the spike is detectable following the addition of the milk.
Heating the spiked samples to 65 C raises the levels somewhat on average, but this increase in activity is at least partially due to the release of endogenous alkaline phosphatase enzyme.
Table 1: Measurement of SEAP activity in cows, milk.
Sample Endogenous Heated Spiked Spiked/heated Cow 1 52 60 282 307 Cow 2 59 62 198 190 Legend: Cows' milk was processed as described in the text. SEAP activities are given in 1000's of luminometer units. "Endogenous" refers to the endogenous background activity present in the aqueous phase of the milk; "Heated" refers to the background activity in the milk following a 30 minute incubation at 65 C; "Spiked" refers to the unheated milk following the addition of the SEAP spike; "Spiked/heated"
represents the spiked sample following a 30 minute incubation at 65 C. The SEAP spike consisted of 1,075 (x1000) units.
Increasing the level of the alkaline phosphatase-inhibitor homoarginine in the samples had no effect on the SEAP activity measurements. On the other hand, acid curdling of the milk to remove caseins, a standard method used to fractionate milk, resulted in the loss of the bulk of the SEAP spike.
Figure 7 shows milk samples with and without the addition of chicken a-amylase preparations. For whole milk, the gel assay shows a light smear throughout the entire lane (see lane 1). This is due to the effect of the abundant milk casein proteins. However, the band of activity corresponding to the chicken amylase reporter 'spike' is clearly visible even in whole milk (lane 2). Our standard protocol for the amylase reconstitution experiments included an acid curdling step to remove caseins from the whole milk. This treatment was incompatible with the SEAP reporter system because it almost entirely depleted the milk aqueous phase of alkaline phosphatase activity (see above).
Following neutralization and centrifugation of the milk containing the chicken a-amylase spike, the samples were concentrated and analyzed by the standard gel assay. The removal of the caseins completely eliminated any endogenous background activity from the milk samples, and left the chicken amylase band clearly visible in the spiked samples (lanes 7&8). Thus, chicken a-amylase is superior to SEAP as a secreted reporter enzyme in biological fluids such as cow's milk.
15 The present invention has been described with regard to preferred embodiments.
However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described in the following claims.
- .._.. ... r.,~~, ~,. <..r, . ~,~,.,. ,...~ .
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Her Majesty in Right of Canada as Represented by Agriculture and Agri-Food Canada (B) STREET: Central Experimental Farm (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1A 0C6 (A) NAME: The University of Ottawa (B) STREET: 115 Seraphin Marion (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1N 6N5 (ii) TITLE OF INVENTION: Secreted alpha-Amylase as a Reporter Gene (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM:
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(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Her Majesty in Right of Canada as Represented by Agriculture and Agri-Food Canada (B) STREET: Central Experimental Farm (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1A 0C6 (A) NAME: The University of Ottawa (B) STREET: 115 Seraphin Marion (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1N 6N5 (ii) TITLE OF INVENTION: Secreted alpha-Amylase as a Reporter Gene (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs (B) TYPE: riucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs (B) TYPE: riucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Claims (18)
1. A secreted reporter gene system comprising a DNA sequence encoding:
a signal peptide;
a chicken .alpha.-amylase protein encoded by the DNA sequence of SEQ ID NO.: 1 and;
a transcription termination region, wherein said system is for use in mammalian cells.
a signal peptide;
a chicken .alpha.-amylase protein encoded by the DNA sequence of SEQ ID NO.: 1 and;
a transcription termination region, wherein said system is for use in mammalian cells.
2. The reporter gene system of Claim 1, wherein the signal peptide is derived from a Drosophila melanogaster .alpha.-amylase gene.
3. The reporter gene system of Claim 2, wherein the Drosophila melanogaster .alpha.-amylase gene is from the Oregon-R strain.
4. The reporter gene system of Claim 3, wherein the signal peptide is encoded by the DNA sequence of SEQ ID NO: 5.
5. The reporter gene system of Claim 1, wherein the transcription termination region is derived from a Drosophila melanogaster .alpha.-amylase gene.
6. The reporter gene system of Claim 5, wherein the Drosophila melanogaster .alpha.-amylase gene is from the Oregon-R strain.
7. The reporter gene system of Claim 6, wherein the transcription termination region is encoded by the DNA sequence of SEQ ID NO.: 2.
8. The reporter gene system of Claim 1, wherein said system further comprises a promoter region.
9. The reporter gene system of Claim 8, wherein the promoter region is selected from the group consisting of: a Cytomegalovirus promoter, a murine retroviral promoter and a rat beta-actin promoter.
10. The reporter gene system of Claim 9, wherein the promoter region is a Cytomegalovirus promoter.
11. The reporter gene system of Claim 8, wherein the signal peptide is derived from a Drosophila melanogaster .alpha.-amylase gene.
12. The reporter gene system of Claim 11, wherein the Drosophila melanogaster .alpha.-amylase gene is from the Oregon-R strain.
13. The reporter gene system of Claim 12, wherein the signal peptide is encoded by the DNA sequence of SEQ ID NO.: 5.
14. The reporter gene system of Claim 8, wherein the transcription termination region is derived from a Drosophila melanogaster .alpha.-amylase gene.
15. The reporter gene system of Claim 14, wherein the Drosophila melanogaster .alpha.-amylase gene is from the Oregon-R strain.
16. The reporter gene system of Claim 15, wherein the transcription termination region is encoded by the DNA sequence of SEQ ID NO.: 2..
17. A secreted reporter gene system comprising a DNA sequence encoding:
a signal peptide derived from a Drosophila melanogaster .alpha.-amylase gene;
a chicken .alpha.-amylase protein encoded by the DNA sequence of SEQ ID NO.:
1; and a transcription termination region derived from a Drosophila melanogaster .alpha.-amylase gene, wherein said system is for use in mammalian cells.
a signal peptide derived from a Drosophila melanogaster .alpha.-amylase gene;
a chicken .alpha.-amylase protein encoded by the DNA sequence of SEQ ID NO.:
1; and a transcription termination region derived from a Drosophila melanogaster .alpha.-amylase gene, wherein said system is for use in mammalian cells.
18. A secreted reporter gene system comprising a DNA sequence encoding:
a Cytomegalovirus promoter, a signal peptide derived from a Drosophila melanogaster .alpha.-amylase gene;
a chicken .alpha.-amylase protein encoded by the DNA sequence of SEQ ID NO.:
1; and a transcription termination region derived from a Drosophila melanogaster .alpha.-amylase gene, wherein said system is for use in mammalian cells.~
a Cytomegalovirus promoter, a signal peptide derived from a Drosophila melanogaster .alpha.-amylase gene;
a chicken .alpha.-amylase protein encoded by the DNA sequence of SEQ ID NO.:
1; and a transcription termination region derived from a Drosophila melanogaster .alpha.-amylase gene, wherein said system is for use in mammalian cells.~
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2203613 CA2203613C (en) | 1997-04-24 | 1997-04-24 | Secreted .alpha.-amylase as a reporter gene |
| AU60856/98A AU6085698A (en) | 1997-04-24 | 1998-02-26 | Secreted alpha-amylase as a reporter gene |
| PCT/CA1998/000157 WO1998049320A1 (en) | 1997-04-24 | 1998-02-26 | SECRETED α-AMYLASE AS A REPORTER GENE |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2203613 CA2203613C (en) | 1997-04-24 | 1997-04-24 | Secreted .alpha.-amylase as a reporter gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2203613A1 CA2203613A1 (en) | 1998-10-24 |
| CA2203613C true CA2203613C (en) | 2008-01-15 |
Family
ID=4160506
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2203613 Expired - Fee Related CA2203613C (en) | 1997-04-24 | 1997-04-24 | Secreted .alpha.-amylase as a reporter gene |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU6085698A (en) |
| CA (1) | CA2203613C (en) |
| WO (1) | WO1998049320A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6838239B1 (en) | 1999-10-13 | 2005-01-04 | San Diego State University Foundation | Chitobiase as a reporter enzyme |
| WO2001027322A2 (en) * | 1999-10-13 | 2001-04-19 | Elitra Pharmaceuticals, Inc. | Chitobiase as a reporter enzyme |
| US6620585B1 (en) | 2000-08-02 | 2003-09-16 | Elitra Pharmaceuticals, Inc. | Use of ectoenzymes and secreted enzymes to monitor cellular proliferation |
| DK1644485T3 (en) * | 2003-07-08 | 2011-08-15 | Axiogenesis Ag | Secreted proteins as markers of cell differentiation |
| GB0322196D0 (en) * | 2003-09-23 | 2003-10-22 | Cxr Biosciences Ltd | Excretable reporter systems |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05184352A (en) * | 1990-01-16 | 1993-07-27 | Centro De Ing Genetica Y Biotecnol | Method for expressing dissimilar gene in pichia pastoris yeast, expression vector and transforming microorganism |
-
1997
- 1997-04-24 CA CA 2203613 patent/CA2203613C/en not_active Expired - Fee Related
-
1998
- 1998-02-26 WO PCT/CA1998/000157 patent/WO1998049320A1/en active Application Filing
- 1998-02-26 AU AU60856/98A patent/AU6085698A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1998049320A1 (en) | 1998-11-05 |
| CA2203613A1 (en) | 1998-10-24 |
| AU6085698A (en) | 1998-11-24 |
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