Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02203613 2004-12-29
-1-
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.
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
CA 02203613 2004-12-29
-2-
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) .
CA 02203613 1997-04-24
-3-
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
CA 02203613 1997-04-24
-4-
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
CA 02203613 1997-04-24
-5-
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.
CA 02203613 1997-04-24
-6-
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.
CA 02203613 1997-04-24
-7-
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
CA 02203613 1997-04-24
-8-
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
CA 02203613 1997-04-24
-9-
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:
179-
187). Hybridization was carried out under conditions of reduced stringency at
37 C
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
CA 02203613 2004-12-29
-10-
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 ..>. ..
..,...,_.__...._... . .._....._.__ _ .. .__.........-.._._.. ~ _. . ......_...
. ..__...._._. ... __ ..
CA 02203613 1997-04-24
-11-
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:
CA 02203613 1997-04-24
-12-
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-
CA 02203613 1997-04-24
-13-
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
CA 02203613 1997-04-24
-14-
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),
MA104
(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
CA 02203613 1997-04-24
-15-
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
CA 02203613 1997-04-24
-16-
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
CA 02203613 2004-12-29
-17-
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, . ~,~,.,. ,...~ .
CA 02203613 1997-07-22
-18-
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:
(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:
(A) APPLICATION NUMBER: 2,203,613
(B) FILING DATE: April 24, 1997
(C) CLASSIFICATION:
(2) INFORMATION FOR SEQ ID NO: 1:
CA 02203613 1997-07-22
-19-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1505 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GCTAGCTCAG TACAATCCCA ACACTCAGGC TGGGAGGACA TCTATCGTGC ATCTCTTTGA 60
ATGGCGCTGG GCCGACATTG CACTGGAGTG CGAACACTAT TTAGCTCCTA ATGGGTTTGG 120
AGGAGTTCAG GTTTCTCCTC CAAATGAAAA CATTGTCATT ACTAATCCGA ACAGGCCCTG 180
GTGGGAAAGA TACCAGCCCA TCAGCTACAA GATCTGCAGT CGATCGGGCA ATGAAAATGA 240
ATTCAGAGAC ATGGTGACCA GATGCAACAA TGTTGGAGTT CGTATTTATG TGGATGCTGT 300
TGTCAATCAC ATGTGTGGAT CTATGGGTGG CACGGGCACC CACTCAACAT GTGGGAGCTA 360
TTTCAACACC GGGACTAGAG ATTTTCCCGC TGTGCCGTAC TCTGCCTGGG ATTTCAATGA 420
CGGCAAATGT CACACTGCAA GTGGAGACAT CGAAAATTAT GGGGACATGT ATCAGGTCCG 480
GGATTGCAAG TTGTCCAGCC TTCTTGATCT GGCTCTGGAG AAGGACTATG TACGCTCAAC 540
AATTGCAGCG TACATGAATC ACCTCATTGA TATGGGTGTA GCAGGGTTCC GGATCGATGC 600
TGCCAAGCAT ATGTGGCCAG GGGACATAAG AGCGTTTCTG GACAAACTGC ACGATCTAAA 660
TACTCAGTGG TTTTCAGCAG GAACGAAACC CTTTATTTAC CAAGAGGTAA TTGACTTGGG 720
AGGAGAGCCA ATCACAGGCA GTCAGTACTT TGGGAATGGC CGCGTGACAG AATTCAAGTA 780
CA 02203613 1997-07-22
-20-
TGGTGCCAAA CTGGGGACGG TGATCCGGAA GTGGAATGGA GAGAAGATGG CCTACTTAAA 840
GAACTGGGGA GAAGGCTGGG GCTTTGTGCC TTCTGACAGA GCCCTGGTGT TTGTGGATAA 900
CCACGACAAC CAGCGGGGGC ACGGGGCAGG CGGAGCTTCC ATTCTTACTT TCTGGGATGC 960
CAGGCTTTAT AAAATGGCGG TTGGTTTCAT GCTCGCTCAT CCGTACGGGT TCACACGGGT 1020
GATGTCAAGT TATCGTTGGC CAAGATATTT CGAAAACGGA GTGGATGTTA ACGACTGGGT 1080
GGGACCACCA AGTAACTCGG ACGGATCGAC GAAGTCCGTT ACAATCAACG CAGACACTAC 1140
CTGTGGCAAT GACTGGGTCT GCGAACATCG CTGGCGACAA ATAAGGAACA TGGTTATCTT 1200
CCGTAATGTG GTAGACGGTC AGCCTTTCTC AAACTGGTGG GACAACGGGA GCAATCAAGT 1260
AGCTTTCGGT CGCGGCGACA GAGGCTTCAT TGTCTTTAAT AATGATGACT GGTATATGAA 1320
TGTCGATTTG CAAACTGGTC TGCCTGCTGG AACCTACTGC GATGTTATTT CTGGACAAAA 1380
GGAAGGCAGT GCGTGTACTG GAAAGCAGGT GTACGTTTCC TCGGATGGAA AGGCCAATTT 1440
CCAGATTAGT AACAGCGATG AAGATCCATT TGTTGCAATT CACGTTGATG CCAAGTTATA 1500
AGCTT 1505
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 568 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
CA 02203613 1997-07-22
-21-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AAGCTTGTAA ACAGCTGGGG AGCATGGCGA ACAGCCAGGC AATTAATTGA GATTATTAAT 60
TGTACGAAAT ATATATGATG AGATTATAAA CACACAACAC TTTTATTCGC AAGGGATGAT 120
AAGAATCTAA TATATATATT ATCTGGGCTT CAAAGCATTG ATTTTATTTA TTGAGTCAAG 180
AGGGAAATTT ATTTTCTTGT TATTCTCTGT CCAGGTCTAA AGTCCCGAGC GGTGAGGCTA 240
TCTATTGATT TGGACATTCC AATCGAATAC AAAACAGAGA TACAGAAATT TGCGAAAAAA 300
TTTGATAACA ATCGTGGATT TTACGAATTA GACAAATTGA TATGTGCTTG CTAATTGATG 360
TGGCATGAAA TAAGAAATTT ATAAGGACGT TTTCAAGTGC TTCTATTTTA AACATTCAGG 420
ATTTTTTTTT AAAGCAGACA GCTTTCAACA GGTTTGATGA GAATTTGAAT ATTGATTGTT 480
GACTTTAGCT ATACATAAAT CACACCTCAT CCACCCATTG TGGTATCCTT CGAAGGACTT 540
GGGAACTGGA TCCTCTAGAA GGCGCGCC 568
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ATGCTAGCTC AGTACAATCC CAACACTCAG GCT 33
CA 02203613 1997-07-22
-22-
(2) INFORMATION FOR SEQ ID NO: 4:
(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:
CGAAGCTTAT AACTTGGCAT CAACGTGAAT TG 32
(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:
CCATGGTTCT GGCCAAGAGC ATAGTGTGCC TCGCCCTCCT GGCGGTGGCG CTAGCT 56
