Download Automated sorting of live transgenic embryos

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RESEARCH ARTICLES
Automated sorting of live
transgenic embryos
© 2001 Nature Publishing Group http://biotech.nature.com
Eileen E.M. Furlong, David Profitt, and Matthew P. Scott*
Departments of Developmental Biology and Genetics, Howard Hughes Medical Institute, Beckman Center B300, 279 Campus Drive,
Stanford University School of Medicine, Stanford, CA 94305-5329. *Corresponding author ([email protected]).
Received 7 August 2000; accepted 10 November 2000
The vast selection of Drosophila mutants is an extraordinary resource for exploring molecular events
underlying development and disease. We have designed and constructed an instrument that automatically separates Drosophila embryos of one genotype from a larger population of embryos, based on a fluorescent protein marker. This instrument can also sort embryos from other species, such as
Caenorhabditis elegans. The machine sorts 15 living Drosophila embryos per second with more than 99%
accuracy. Sorting living embryos will solve longstanding problems, including (1) the need for large quantities of RNA from homozygous mutant embryos to use in DNA microarray or gene-chip experiments,
(2) the need for large amounts of protein extract from homozygous mutant embryos for biochemical
studies, for example to determine whether a multiprotein complex forms or localizes correctly in vivo
when one component is missing, and (3) the need for rapid genetic screening for gene expression
changes in living embryos using a fluorescent protein reporter.
Keywords: Drosophila, embryo, development, green fluorescent protein
To fully benefit from the remarkably comprehensive new information about the Drosophila and C. elegans genomes, it would be a
tremendous advantage to be able to isolate large quantities of
mutant embryos and embryo populations at defined developmental
stages. The combination of nearly a century of mutant collection
and analysis, in concert with new technologies such as DNA
microarrays, provide an opportunity to understand genetic networks, reveal regulatory pathways, and detect responses to environmental perturbations. Of the estimated 13,601 Drosophila genes,
about 3,600–4,000 are expected to be essential for survival.
Thousands of existing lethal mutations are kept as stocks of heterozygous flies. One quarter of the embryos produced by such a
population will be homozygous for the mutation of interest1. The
threefold excess of embryos carrying the wild-type gene, often on a
balancer chromosome, creates problems for many types of biochemical and molecular experiments (Fig. 1A).
To overcome this problem, we have designed, built, and tested an
instrument that sorts embryos of a particular genotype according to
their levels of fluorescence. The machine was designed for use with
Drosophila embryos, but the same principles can be applied to sorting
embryos from other species, such as C. elegans, that have been engineered to contain fluorescent cells. We initially developed the machine
for Drosophila because of the available genetic tools and the recent near
completion of the whole-genome sequencing project.
To ensure that lethal mutations of interest are inherited by the
next generation, Drosophila geneticists use balancer chromosomes.
These chromosomes are multiply inverted to minimize recombination events, and often carry lethal mutations so that homozygous
balancer embryos die. The homozygous mutant embryos also die, so
the only embryos that survive into adulthood are the heterozygous
mutant/balancer embryos (Fig. 1A). Adults containing a balancer
chromosome produce 25% homozygous embryos, the embryos that
most people would like to study. A number of fly strains have a gene
encoding green fluorescent protein (GFP) inserted into the balancer
chromosome2. It is therefore possible to distinguish, in living
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embryos, homozygous mutant embryos that lack GFP expression
from heterozygous and homozygous-balancer embryos that have
GFP (Fig. 1A). This paper describes a machine that can sort
Drosophila embryos based on the presence or absence of GFP. The
machine can separate the 25% homozygous mutant embryos from
the 75% that contain the GFP-balancer.
Cell sorting is similar in basic principles to embryo sorting.
Fluorescence-activated cell sorting (FACS) is widely used to isolate
subpopulations of cells based on antigen display, nucleic acid content,
and gene expression3,4. During FACS analysis, cells are observed in
laminar flow, which is then dispersed into droplets of solution such
that only one cell is contained within a droplet5. The cells are sorted by
electrostatic deflection of the droplet, displacing it away from the laminar flow of solution. Whole embryos are too large to be sorted using
conventional FACS technology. Drosophila embryos are 450–500 µm
long and 150–200 µm wide6 and C. elegans first-instar larvae (L1) are
250 µm long7; a typical mammalian cell has a diameter of 10–20 µm.
Results
Mechanics of the embryo sorter. An overview of the instrument
(Fig. 1B) outlines how it operates. Collected embryos are maintained suspended in buffer in a chamber using a magnetic stirrer. A
peristaltic pump continuously moves solution through the chamber
into an optical cuvette (Fig. 2A). Solution enters and exits the chamber at a fixed rate (∼6 ml/min). Some of the suspended embryos randomly enter the flow of liquid that is exiting the chamber. This flow
carries the embryos through the optical cuvette, which consists of a
0.4 mm square glass tube embedded in a holder containing immersion oil. The diameter of the tube restricts the embryos to two possible orientations (Fig. 2B).
Embryos passing through the cuvette are illuminated by an argon
laser (488 nm). Light emitted from an embryo is detected perpendicular to the laser excitation light (Fig. 2C). The emitted light passes through an aspheric condenser lens that collects and collimates
the light and then through a plano-convex focusing lens followed by
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RESEARCH ARTICLES
A
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B
Figure 1. Sorting embryos based on their expression of GFP.
(A) Drosophila lethal mutations are maintained over a balancer
chromosome. Balancer chromosomes are multiply inverted
chromosomes, to minimize recombination events, that often carry
lethal mutations. They allow the stable maintenance of recessive lethal
mutations, because the only embryos that can survive to adulthood are
the heterozygous embryos (mutation/balancer). However, for any
embryo collection from these parents, only 25% of the embryos contain
the homozygous mutation of interest. Using a GFP marker gene on the
balancer chromosome, it is possible to distinguish, in living embryos,
between this minority population of mutant embryos and their
balancer-containing siblings. (B) Schematic diagram of the embryo
sorter outlines how the machine operates. Suspended embryos
randomly enter the fluid flow through the optical cuvette, where they
are illuminated by an argon laser (488 nm). The emitted light is detected
at three points along the light path: (1) a diode, (2) photomultiplier tube
1 (PMT-1), which detects the light between the 510 ± 5 nm (GFP), and
(3) PMT-2, which detects all the light that passes through the 520 nm
dichroic mirror (autofluorescence). If the GFP peak is above or below a
defined threshold, a signal is sent to the mechanical switch to direct the
embryo flow to either the save or waste tube.
two dichroic mirrors (optical cutoff at 498 nm and 520 nm)
(Fig. 2D). Light is detected at three points along the light path: (1) A
diode, measuring light that has passed through the lenses, detects all
interruptions of the light. The diode signal, marking the position of
each passing embryo, is analyzed for amplitude peaks by a dedicated
microcontroller. (2) A photomultiplier tube (PMT-1) detects the
light between the 498 nm and 520 nm dichroic mirrors. This light
passes through a narrow filter (510 ± 5 nm) before reaching the
PMT-1 and therefore detects mainly the GFP signal. (3) PMT-2
detects light that passes through the 520 nm dichroic mirror. This
light passes through a 515–60 nm barrier filter before reaching the
PMT-2, which detects the background fluorescence or autofluorescence emanating from the yolk of the embryo. When the diode
detects an embryo, the microcontroller measures the voltage detected for the GFP (VGFP), autofluorescence (Vauto), and threshold signal
(Vth) from the PMTs. The threshold signal is chosen by the user and
is the level of GFP above or below which the machine should save an
embryo. The computer draws peaks for each embryo, one for the
GFP level and a second for autofluorescence. To obtain GFPcontaining embryos, the microcontroller evaluates the condition
VGFP – Vauto > Vth. If the condition is true, the signals “New embryo
detected” and “Save” are generated. If the condition is false, the signal “New” is the only output. Conversely, to obtain embryos that do
not contain any GFP, a condition VGFP – Vauto > Vth “false” would generate a “New embryo detected” and a “Save” output. The “New” and
“Save” outputs are sent to electromagnets that control the switch
that directs embryos into one tube or another.
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Figure 2. Optical system and the mechanical switch used in the
embryo sorter. (A) Embryos pass through an optical cuvette
composed of a 0.4 mm diameter square tube, enclosed in a square
chamber. (B) Schematic diagram of the 0.4 mm square tube. The
diameter of the tube restricts the number of possible orientations of
the embryos. (C) The optical cuvette is aligned with the optics
chamber, ensuring appropriate alignment of the viewing windows so
emitted light from the embryo passes through the focusing lens to the
PMTs. (D) Side view of the inside of the optics chamber. The emitted
light (red arrow) passes through this chamber and is detected by the
diode and the PMTs. (E) End-on view of the optics cuvette shown in
(A). The embryos exit the central hole in a droplet of solution before
reaching the switch. (F) The end of the cuvette shown in (E) attaches
to a metal fluid ring. The fluid ring has an inflow of solution at high
speed; the drop of solution containing the embryo enters a sheath of
liquid and exits through the central hole in a stream of liquid. (G) View
of the embryo switch from above. This switch is aligned underneath
the cuvette-fluid ring, underneath the blue arrow in (C). (H) Side view
of the embryo switch. The default position is for the switch to be
aligned to the waste tube; the switch moves to the right to collect a
Save embryo. (Yellow arrows indicate direction of fluid and embryo
flow. Red arrows indicate direction of the light path.)
The switch is composed of a waste tube and a save tube, separated by a very thin central wall (Fig. 2G, H). The embryo and solution
flow is always aligned to the waste tube, which is the default position.
The two tubes are attached to a rare-earth neodymium supermagnet suspended between two electromagnets. Electric current
applied in one direction to the electromagnets exerts force on the
suspended magnet in one direction. Reversing the current produces
a force in the opposite direction. The magnet moves the switch to the
left or right, that is, to the “Save” or “Waste” position.
The embryo–buffer suspension passes through the optical cuvette
at a rate of ∼6 ml/min. Buffer containing the embryos drips from the
end of the optical cuvette into the switching mechanism. To facilitate
rapid and accurate switching, the drop containing the embryo enters
a high-speed fluid stream from a second pump (Fig. 2F). The
mechanical switch is aligned underneath the high-speed stream of
liquid to collect the embryos (below the blue arrow in Fig. 2C).
The timing of the embryo-sorting switch is controlled by a second dedicated microcontroller that receives the Save and New outputs from the optics microcontroller. For any fluid flow rate there is
a predictable time from embryo detection to the arrival of the
embryo at the sorting switch. For example, with a fluid flow of
∼6 ml/min there is an 11–12 ms delay from the time of embryo
detection to the time when the embryo reaches the switch. If a Waste
embryo is ahead of a Save embryo, sufficient time must be allowed
for the Waste embryo to pass through the switch before the switch
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B
Figure 3. Viability of sorted embryos. One-hour embryo collections
were obtained at three stages of development. Each embryo
collection was divided into four groups of embryos that were treated
as follows: (1) untreated embryos, (2) dechorionated embryos,
(3) dechorionated embryos that were placed in sorting buffer for 1 h,
and (4) dechorionated embryos that were sorted in the machine for
1 h. After 1 h the embryos were placed in a food container and
allowed to develop at 25°C. After two weeks the flies that had
eclosed in each vial were counted. This experiment was conducted
three independent times for each of the four conditions at three
developmental stages.
moves to collect the Save embryo. Conversely, if a Save embryo is
ahead of a Waste embryo, the switch must return to the Waste position before the second embryo arrives. Timing is accomplished by a
software algorithm that effectively maps the position of the embryo
in real time as it travels from the detector to the sorting switch.
A detailed description of how to build the sorter is available as
supplementary information (in the Web Extras page of Nature
Biotechnology Online).
Viability of sorted embryos. We determined what effect sorting
has on the viability of the embryos. Three independent embryo collections from three different stages in development were analyzed.
The fly strain was wild type (Canton S). Each embryo collection was
passed through the embryo sorter for 1 h. The embryos were counted and allowed to develop to adulthood at 25°C. After 14 days, >93%
of the embryos had reached adulthood (Fig. 3). No significant difference was observed between sorted embryos and those that were
held in sorting buffer for 1 h or just dechorionated. Any deleterious
effects of sorting are evidently due to incubating the embryos in
buffer or to dechorionation, rather than to the machine itself.
Sorting speed for transgenic GFP-bearing embryos. The optimal
speed of embryo sorting is determined both by the machine’s theoretical sorting speed and by the density of the embryos that will be
sorted. The mechanical switch in the embryo sorter can open and
close in 10 ms, giving a theoretical sorting speed of approximately
100 embryos/s. However, operating the machine at this speed would
greatly reduce the efficiency of sorting. An adequate time interval
must be preserved between Waste and Save embryos in order to
ensure that a Waste embryo has passed through the switch before
flipping the switch to collect a Save embryo. The instrument is set so
that if a Waste embryo is too close to a Save embryo (that is, within
10 ms), both embryos are sent to the waste tube. A lower embryo
density results in greater spacing between embryos, therefore reducing the probability of two embryos being within 10 ms of each other.
Similar logic applies to FACS sorting.
Empirically, the optimal sorting rate is 15 embryos/s. To maintain a
constant embryo density, the embryo sorter uses a twochamber system: a high-density embryo chamber, and a low-density
embryo chamber. Freshly collected embryos are added to the highdensity chamber. The computer monitors the total number of detected
embryos, the number of saved embryos, and the rate of embryo flow
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C
Figure 4. Verification of sorting. (A) The panel on the left contains a
typical embryo collection from the Kr-GFP balancer strain of
Drosophila2. This mixed population of GFP-containing embryos can
be sorted to obtain non-GFP-expressing embryos or GFP-expressing
embryos. (B) Immunostains of wild-type and sorted twist mutant
embryos. A molecularly characterized loss-of-function twist allele
was placed in trans to a GFP-balancer chromosome. Embryo
collections from this stock were sorted, selecting for nonGFP-containing embryos (i.e., homozygous twist mutant embryos).
The sorted embryos were formaldehyde-fixed and immunostained
with dMef2 antibody (Drosophila myocyte-enhancing factor). The left
panel shows a wild-type embryo immunostained with anti-dMef2.
The right panel shows an embryo that was sorted selecting for nonGFP-expressing embryos (i.e., homozygous mutants), also
immunostained with dMef2 antibody. The sorted embryo has no
mesodermal staining, verifying that this is a homozygous twist
mutant embryo. (C) The panel on the left contains a mixed population
of GFP and non-GFP C. elegans L4 larvae. This mixed population was
sorted to obtain the population of GFP-containing larvae. To stop the
larvae from moving to take these photographs, the larvae were
heated to 70°C for 10–20 s.
per second. Normally embryos flow from the lowdensity chamber to the cuvette. If the rate of embryo passage falls
below a defined threshold (i.e., 15 embryos/s) the computer sends a
signal to a fluid valve. The valve redirects the fluid flow to pass through
the high-density chamber into the low-density chamber and then to
the optical cuvette. This moves embryos from the high-density chamber to the low-density chamber. Maintaining the embryo density in
this way permits the embryos to be efficiently sorted at a constant rate.
Using the optimal sorting speed, we have routinely separated
13,000 homozygous embryos from a population of ∼39,000
embryos in 1 h.
Verification of sorting. The accuracy of sorting was determined
by examining the Saved embryo collections with a GFP microscope
(Leica MZ12 GFP plus). Mixed populations of Drosophila embryos
were sorted, selecting for embryos with low light intensity detected
by the GFP-PMT (Fig. 4A). The population of “non-GFP” embryos
had no visible GFP, or fewer than 1% of the embryos had very low
levels of GFP. Embryos can also be selected for high GFP-PMT
intensities, corresponding to GFP-containing embryos (Fig. 4A).
The intensity of GFP can vary from embryo to embryo. Within a
1 h staged collection, younger embryos in the population may be
just initiating GFP expression, making it possible to mistake a GFPexpressing embryo for a homozygote. To more precisely determine
the accuracy of sorting, we used a genetic marker. Homozygous
twist mutants have a well-documented phenotype. Twist is essential
for mesoderm development; homozygous twist mutant embryos
have no mesoderm8,9. The phenotype of homozygotes is dramatic
and easily detected by immunostaining (Fig. 4B).
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Embryos were collected and sorted from a fly stock carrying a
loss-of-function twist allele in trans to a GFP-balancer chromosome.
Non-GFP containing embryos (i.e., homozygous twist mutant
embryos) were formaldehyde-fixed10 and immunostained with an
antibody that recognizes the mesoderm (Fig. 4B). Sorted immunostained embryos were examined under a microscope to count
embryos that looked normal (i.e., were not homozygous twist
mutants) and therefore were sorting errors. Only 15 out of 1,700
sorted embryos were wild type, indicating an error rate of 0.88%.
Therefore the machine operates at >99% accuracy. This accuracy
was not reduced when the embryos were sorted at a rate higher than
15 embryos/s, although as noted above the efficiency went down.
Mixed populations of C. elegans L4 larvae were also successfully
sorted, selecting larvae that contain GFP (Fig. 4C). The larvae were
highly motile after sorting, indicating that the sorting process had
little or no effect on viability. The machine functions between 80 and
99% accuracy when sorting C. elegans. The variability in the sorting
accuracy is due to the size differences between C. elegans L4 larvae
and Drosophila embryos. Our machine was scaled for use with
Drosophila embryos. The optical cuvette was manufactured so that
only one Drosophila embryo can pass through the optical cuvette at a
time. Caenorhabditis elegans L4 larvae are narrower than Drosophila
embryos (Drosophila embryos being 150–200 µm wide and C. elegans L4 larvae 50–80 µm wide), and they can sometimes pass each
other while traversing the optical cuvette between the point of detection and the point of sorting. The accuracy for C. elegans sorting can
readily be improved by manufacturing smaller optical cuvettes and
identifying the optimal size.
Discussion
Applications of the embryo sorter. An important use of the embryo
sorter is to separate homozygous Drosophila mutant embryos from
their GFP-balancer siblings. At present the molecular and biochemical analysis of intact homozygous mutant embryos requires manual
sorting of thousands of embryos in order to separate mutants from
the 75% of the embryos that contain a balancer chromosome. As a
result of the time-consuming and laborious task of hand sorting
mutant embryos, biochemical and molecular biology analyses of
Drosophila mutant embryos have been severely restricted.
Embryonic development occurs very rapidly (22 h in Drosophila and
15 h in C. elegans), making it impossible to hand sort large quantities
of embryos within a narrow window of time. Automatic sorting of
15 living embryos per second with more than 99% accuracy makes
possible many previously difficult experiments.
Large quantities of mutant embryos will facilitate whole-genome
gene expression analyses of mutants using DNA microarrays. For a
typical Drosophila complementary DNA (cDNA) microarray experiment, we currently make a fluorescent cDNA probe from 2.5–4 µg
of polyA+ RNA. This amount of RNA is purified from ∼3,000–4,000
embryos, a number easily obtained with the sorter. These studies
will yield insights into molecular ramifications of losing a gene function. For example, embryos that are homozygous mutant for a transcription factor can be isolated and the messenger RNA (mRNA)
used in microarray experiments to identify direct and indirect transcriptional targets. Although many transcription factors are known,
few of the genes they regulate have been identified. A much more
complete analysis of gene regulatory relationships will yield extensive insights into cell fate determination, organogenesis, oncogenesis, and cell proliferation mechanisms. Homozygous mutant
embryos can also be used for biochemical studies to analyze the ability of proteins to form multisubunit complexes or to have a normal
subcellular localization when one member of a complex is absent.
The sorter will be useful for selecting GFP-containing embryos.
More than 93% of the embryos that pass through the embryo sorter
survive to adulthood, so the sorter can be used to carry out genetic
156
screens using changes in the production of a fluorescent protein to
reveal a mutation. For example, genetic screens could be conducted
to identify new oncogenes by isolating embryos with more GFP as a
result of an increased number of GFP-expressing cells. The sorted
embryos develop into adults, allowing the maintenance of a new
strain of flies of the desired genotype.
Usefulness of multiple fluorescent protein tags. At present, we
are using GFP-labeled embryos. Other fluorescent proteins, including red fluorescent protein (RFP), cyan fluorescent protein (CFP),
and blue fluorescent protein (BFP)11, can be used to do two-color
embryo sorting using appropriate filter sets. For example, to select
embryos that contain CFP but not GFP, the machine can separate
non-GFP embryos from GFP-expressing embryos. After changing
filters, the selected non-GFP embryos can be sorted according to
CFP content. In the future, additional filter sets and a third PMT can
be added to detect both fluorescent channels simultaneously.
As more enhancers become characterized, it may be possible to
create a set of Drosophila and C. elegans strains that each express a
fluorescent protein at a specific stage of development. This would
allow stage-specific embryo sorting. Any variability in microarray
gene expression profiles that result from slight staging differences
would be greatly reduced.
Experimental protocol
Drosophila embryos have an outer membrane, the chorion, which is often
removed, without harming the embryos, to significantly enhance the GFP
intensity. The chorion is removed by placing the embryos in 50% bleach for
2 min and then washing the embryos through a sieve into a solution of
PBS/2% Tween-20. All of the sorting is conducted in this buffer. The Tween
does not affect viability (Fig. 4) and greatly reduces clumping of the embryos.
The embryos are placed into the high-density embryo chamber. Once all the
air bubbles have been flushed out of the system, some embryos are added to
the low-density embryo chamber and sorting begins. Caenorhabditis elegans
larvae were washed off agar plates in PBS and collected by centrifugation.
Bacteria were removed by washing four times in PBS. The larvae were sorted
in PBS/0.1% Tween-20. A detailed description of how to build the sorter is
available as supplementary information.
Note: Supplementary information can be found on the Nature
Biotechnology website in Web Extras (http://biotech.nature.com/
web_extras).
Acknowledgments
We are grateful to Dr. Stephen Smith for his suggestions and discussions on the
optics used in the machine. We thank Dr. Roel Nusse for his careful reading of the
manuscript, and Dr. Allan Spradling for advice. The C. elegans larvae were
kindly provided by Drs. Peter J. Roy and Stuart Kim. E.F. was supported by a
European Molecular Biology Organization fellowship and a Stanford Berry
Fellowship. The research was supported by the Howard Hughes Medical Institute
and DARPA grant number N00014-98-1-0689.
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