U.S. patent application number 11/048101 was filed with the patent office on 2006-08-03 for methods for staining cells for identification and sorting.
Invention is credited to Amy L. Anderson, Christopher R. Knutson, Daniel Mueth, Joseph Plewa, Evan Tanner.
Application Number | 20060172315 11/048101 |
Document ID | / |
Family ID | 36757022 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172315 |
Kind Code |
A1 |
Anderson; Amy L. ; et
al. |
August 3, 2006 |
Methods for staining cells for identification and sorting
Abstract
The present invention provides novel methods of cell staining,
such as bovine sperm, using electroporation or osmolality
treatments at viability-enhancing temperatures. Furthermore,
methods of highly efficient cell sorting that are especially
suitable in sorting bovine sperm using novel cell staining
procedures are also provided.
Inventors: |
Anderson; Amy L.; (Prospect
Heights, IL) ; Knutson; Christopher R.; (Chicago,
IL) ; Mueth; Daniel; (Chicago, IL) ; Plewa;
Joseph; (Park Ridge, IL) ; Tanner; Evan;
(Chicago, IL) |
Correspondence
Address: |
AKERMAN SENTERFITT
801 PENNSYLVANIA AVENUE N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
36757022 |
Appl. No.: |
11/048101 |
Filed: |
February 1, 2005 |
Current U.S.
Class: |
435/6.14 ;
435/40.5 |
Current CPC
Class: |
G01N 33/689 20130101;
B82Y 5/00 20130101; B82Y 10/00 20130101; G01N 2021/6439 20130101;
G01N 2015/1081 20130101; G01N 1/30 20130101; G01N 15/1425 20130101;
C12N 5/061 20130101; G01N 2015/1006 20130101; G01N 15/14 20130101;
G01N 21/6428 20130101; G01N 2015/149 20130101; A61D 19/04 20130101;
G01N 2015/0065 20130101; C12N 5/0612 20130101; G01N 33/5005
20130101 |
Class at
Publication: |
435/006 ;
435/040.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/48 20060101 G01N033/48 |
Claims
1. A method for staining sperm, comprising: staining sperm with a
dye by mixing the sperm with the dye; and electroporating the sperm
and dye for a period of time to provide substantially uniform
staining and concomitantly to substantially preserve sperm
viability.
2. A method for distinguishing sperm based on DNA content,
comprising: staining sperm with a DNA-selective fluorescent dye by
mixing the sperm with the dye at a temperature substantially equal
to or less than 39.degree. C.; electroporating the sperm and dye
for a period of time to provide substantially uniform staining and
concomitantly to substantially preserve sperm viability; exposing
the sperm to a light source to cause the stained DNA to fluoresce;
detecting a pre-determined fluorescence of the stained DNA, the
pre-determined fluorescence corresponding to DNA content; sorting
the sperm based on the pre-determined fluorescence; and collecting
selected sperm form the sorted sperm.
3. The method of claim 2, wherein the pre-determined fluorescence
corresponds to a desired chromosome, chromosome fragment, an
insertion or a deletion.
4. The method of claim 2, wherein the sperm is from a mammal.
5. The method of claim 4, wherein the mammal is one selected from
the group consisting of bovine, swine, rabbit, alpaca, horse, dog,
cat, ferret, rat, mouse and buffalo.
6. The method of claim 2, wherein the dye is membrane
impermeant.
7. The method of claim 6, wherein the dye comprises at least one
selected from the group consisting of SYTOX blue, SYTOX green,
SYTOX orange, a cyanine dimer, POPO-1, BOBO-1, YOYO-1, TOTO-1,
JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, a cyanine monomer,
PO-PRO-1, BO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3,
LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, acridine
homodimer, 7-amino actinomycin D, ethidium bromide, ethidium
homodimer-1, ethidium homodimer-2, ethidium nonazide, nuclear
yellow and propidium iodide.
8. The method of claim 2, wherein the dye is membrane permeant.
9. The method of claim 8, wherein the dye comprises at least one
selected from the group consisting of SYTO 40 blue-fluorescent
nucleic acid stain, SYTO 41 blue, SYTO 42 blue, SYTO 43 blue, SYTO
44 blue, SYTO 45 blue, a green-fluorescent SYTO dye, SYTO 9 green,
SYTO 10 green, SYTO BC green, SYTO 13 green, SYTO 16 green, SYTO 24
green, SYTO 21 green, SYTO 27 green, SYTO 26 green, SYTO 23 green,
SYTO 12 green, SYTO 11 green, SYTO 20 green, SYTO 22 green, SYTO 15
green, SYTO 14 green, SYTO 25 green, an orange-fluorescent SYTO
dye, SYTO 86 orange, SYTO 81 orange, SYTO 80 orange, SYTO 82
orange, SYTO 83 orange, SYTO 84 orange, SYTO 85 orange, a
red-fluorescent SYTO dye, SYTO 64 red, SYTO 61 red, SYTO 17 red,
SYTO 59 red, SYTO 62 red, SYTO 60 red, SYTO 63 red, a Hoechst dye,
Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, LDS 751 and
dihydroethidium.
10. The method of claim 2, wherein the mixing is at a temperature
between about -4.degree. C. to about 30.degree. C.
11. The method of claim 2, wherein the mixing is at a temperature
about 0.degree. C., 4.degree. C., 12.degree. C. or 30.degree.
C.
12. The method of claim 2, wherein the mixing period of time is
about 1 minute to about 15 minutes.
13. The method of claim 2, wherein the mixing period of time is
less than 1 minute.
14. The method of claim 2, further comprising mixing the sperm with
at least one nanoparticle.
15. The method of claim 2, wherein the nanoparticle comprises a
quantum dot or metallic nanoparticle.
16. The method of claim 2, wherein the nanoparticle comprises a
targeting molecule.
17. The method of claim 2, wherein the targeting molecule binds DNA
or a fluorescent dye.
18. The method of claim 2, further comprising eliminating dead
sperm before sorting the sperm.
19. The method of claim 2, wherein the sperm are sorted by X- or
Y-chromosome DNA content with at least 90% efficiency.
20. The method of claim 2, wherein the viability of the sperm
before sorting is at least 30%.
21. The method of claim 20, wherein the viability of the sperm is
at least 75%.
22. The method of claim 21, wherein the viability of the sperm is
at least 80%.
23. The method of claim 22, wherein the viability of the sperm is
at least 90%.
24. A method for distinguishing sperm based on DNA content and
maintaining sperm viaiblity, comprising: staining sperm with a
DNA-selective fluorescent dye by mixing the sperm with the dye at a
temperature substantially sufficient to maintain a comparatively
high sperm viability rate; electroporating the sperm and dye for a
period of time to provide substantially uniform staining and
concomitantly to substantially preserve sperm viability; exposing
the sperm to a light source to cause the stained DNA to fluoresce;
detecting a predetermined fluorescence of the stained DNA, the
pre-determined fluorescence corresponding to DNA content; sorting
the sperm based on the pre-determined fluorescence; and collecting
selected sperm from the sorted sperm.
25. The method of claim 24, wherein the high sperm viability rate
is at least 70%.
26. A method for distinguishing sperm based on DNA content, wherein
the sperm are stained with a DNA-selective fluorescent dye;
comprising: incubating the sperm under a hypertonic condition and
at a temperature substantially sufficient to maintain a
comparatively high sperm viability rate; transferring the sperm to
a hypotonic condition; exposing the sperm to a light source to
cause the stained DNA to fluoresce; detecting a pre-determined
fluorescence of the stained DNA, the pre-determined fluorescence
corresponding to DNA content; sorting the sperm based on the
pre-determined fluorescence; and collecting selected sperm from the
sorted sperm, wherein the dye is present in a least one of the
hypertonic or hypotonic condition.
27. The method of claim 26, wherein the hypertonic condition is
about less than 250 mOsm and the hypotonic conditions is about
greater than 250 mOsm.
28. The method of claim 27, wherein the incubation temperature is
less than or equal to 4.degree. C.
29. A method for distinguishing sperm based on DNA content while
maintaining sperm viability, comprising: incubating the sperm under
a hypertonic condition to partially dehydrate the sperm, and at a
temperature substantially sufficient to maintain a comparatively
high sperm viability rate; transferring the sperm to a hypotonic
condition; exposing the sperm to a light source to cause the
stained DNA to fluoresce; detecting a pre-determined fluorescence
of the stained DNA, the pre-determined fluorescence corresponding
to DNA content; sorting the sperm based on the pre-determined
fluorescence; and collecting selected sperm from the sorted sperm,
wherein the dye is present in a least one of the hypertonic or
hypotonic condition.
30. The method of claim 29, wherein the high sperm viability rate
is greater than 70%.
31. A method to pre-select the sex of mammalian offspring
comprising: sorting sperm according to the method of claim 2; and
fertilizing an egg obtained from a female animal, the female being
the same species as the male animal which provided the selected
sperm.
32. A method to pre-select the sex of a mammalian offspring
comprising: sorting sperm according to the method of claim 2; and
inseminating a female animal, the female being the same species as
the male animal which provided the selected sperm.
33. A method for staining sperm, comprising permeating the sperm
cell membrane.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application incorporates by reference the related
application, METHOD AND APPARATUS FOR SORTING CELLS, Ser. No.
______, filed ______, and Representative Docket Number
089000-0138.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
[0003] Not Applicable.
FIELD OF THE INVENTION
[0004] The invention relates to techniques and systems for
visualizing live cells using novel staining procedures.
BACKGROUND OF THE INVENTION
[0005] The in vivo identification of a target cell population is
required, and required quickly, in many industries. Such
applications include those where the selected cells are destined
for other applications that require the cells to be living after
identification. For example, cells are processed using
fluorescence-activated cell sorting, where cells are cultured and
expanded in vitro after sorting, or in sperm sorting by gender in
animal husbandry applications.
[0006] Being able to pre-select animal offspring gender would allow
more efficient operations of livestock producers. Dairy farmers
have little use for most bull calves. For example, males are
preferred in beef cattle and sheep because males grow faster,
producing more meat more quickly.
[0007] The male reproductive cells, the sperm, determine the gender
of the offspring. Most males carry an X and a Y sex chromosome,
whereas females carry two X chromosomes. A sperm or an egg contains
one half of that parent's genetic information; however, the egg
only carries an X chromosome one of each pair of autosomes. In
mammals, the egg always contains an X chromosome, while the sperm
carries either an X or Y chromosome.
[0008] Distinguishing male-producing from female-producing sperm is
most easily accomplished by exploiting the difference in the size
of the two sex chromosomes. The X chromosome contains more DNA than
does the Y chromosome. For example, the difference in total DNA
between X-bearing sperm and Y-bearing sperm is 3.4% in boar, 3.8%
in bull, and 4.2% in ram sperm.
[0009] Distinguishing Cells
[0010] To illuminate the workings of cells or distinguish cells
that differ from each other by the slightest difference (e.g.,
expression of a particular molecule), various visualization methods
have been used for decades, from simple light microscopic
observations to high-voltage electronic microscopy. In most of
these techniques, cells or tissue are preserved, usually using a
cross-linking agent such as an aldehyde (proteins, e.g.,
glutaraldehyde and formaldehyde), osmium (lipids) or by
precipitating parts of the cells, such as cold methanol and
proteins. These techniques suffer from the preparation processes
that allow for the visualization. Fixation procedures often incur
artifacts; for example, in the early days of electronic microscopy
(EM), multilamellar bodies were observed but were later understood
to be mostly by-products of the fixation protocols, not actual
structures found in living mammalian cells. While fixation
protocols do preserve some of the cell structure, there are many
structures that are difficult to preserve, or when preserved under
appropriate conditions, the rest of the cell architecture is
destroyed. Classically, this has been the case for the
cytoskeleton, especially for exceptionally dynamic
microtubules.
[0011] To overcome the limitations of visualization techniques in
fixed samples, "in vivo" approaches have been explored. For
example, to understand where native polypeptides localize, those
polypeptides have been purified, associated with a detectable dye
(usually covalently), and then introduced into the cell of interest
and observed (Chamberlain and Hahn, 2000). This approach does offer
the advantages of non-fixed cells; however, the time and expense to
purify a target polypeptide, conjugate it to a dye, and then to
microinject (a task requiring specialized equipment, experience,
skill and patience) the complex into a cell often outweigh the
advantages. Furthermore, only limited numbers of cells could be
examined at any given time due to the limitations of
microinjection.
[0012] With the advent of the discovery of green and other visible
fluorescent proteins (VFPs), however, the ability to visualize
polypeptides--even polypeptide-polypeptide interactions--became
facile and less riddled with artifacts. Green fluorescent protein
is a naturally occurring luminescent protein first found in
jellyfish. Having been cloned, many variants have been produced
that produce a rainbow of colors. In most instances, the protein of
interest is fused by recombinant procedures to a VFP of choice and
the transgene introduced and expressed in the cell of interest
(Chamberlain and Hahn, 2000). While this approach is far superior
to previous methods, many extra, time-consuming, steps are required
from identifying the protein of interest to actually visualizing it
in a living cell.
[0013] Going beyond cellular localization and movement of proteins,
other dyes have been exploited to identify other processes or stain
specific molecules. For example, calcium-mediated signaling is
monitored in living cells using the fura series of dyes. Other
fluorescent dyes have been used to test the molecular size barriers
of gap junctions in, for example, epithelial cells. Finally, other
stains target specific molecules, such as double-stranded
deoxyribonucleic acid (DNA); such stains include some of the
Hoechst series of dyes, propidium iodide and ethidium bromide.
[0014] In each case, however, the challenge of introducing the dye
or stain into a living cells to the appropriate target area is
hindered by the cell membrane which provides a barrier to cells
from the outside world. In many cases, dyes are membrane impermeant
due to their hydrophobic nature or their size; even
membrane-permeant dyes can require long incubation times to breach
the membrane and reach the target molecules or cellular
compartments. Breaching the barrier requires a physical
perturbation of the membrane, such as by microinjection or
fixation.
[0015] Available procedures are few and when available, often face
uncompromising challenges. Even traditional methods of staining DNA
in common methods of sorting sperm cells by gender require
extensive incubation times at elevated temperatures (e.g., 60
minutes at 35.degree. C.; (Johnson, 1992)), permitting quality
degradation of the cells. In addition, staining must be sufficient
so that the signal can be accurately and precisely detected.
SUMMARY OF THE INVENTION
[0016] In a first aspect, the invention discloses methods for
staining cells, such as sperm, including bovine sperm, wherein the
sperm are mixed with a dye of choice and then electroporating them
to facilitate the introduction of the dye. The sperm can be
incubated at temperatures that enhance sperm viability, typically
equal to or less than 39.degree. C.
[0017] In a second aspect, the invention discloses methods for
sorting cells, such as sperm, by distinguishing differences in DNA
content. The cells are stained with a DNA specific dye by mixing
the sperm with the dye and then electroporating them. The sperm can
be maintained at temperatures that enhance sperm viability,
typically equal to or less than 39.degree. C. The sperm are then
passed before an excitation light source causing the stained DNA to
fluoresce, and then passed through means for detecting the
fluorescence and a means for cell sorting, wherein the cells are
sorted by DNA content, and the sorted sperm collected. The methods
and apparatus are appropriate for mammalian sperm sorting, such as
those from bovine, swine, rabbit, alpaca, horse, dog, cat, ferret,
rat, mouse and buffalo. Both membrane permeant and impermeant dyes
can be used. Useful dyes include those from the SYTOX blue, orange
and green series, cyanine dimers and monomers, POPO-1, BOBO-1,
YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3,
PO-PRO-1, BO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3,
LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, acridine
homodimer, 7-amino actinomycin D, ethidium bromide, ethidium
homodimer-1, ethidium homodimer-2, ethidium nonazide, nuclear
yellow, propidium iodide. Other useful dyes include those from SYTO
40 blue, green, orange and red fluorescent dyes, Hoechst dyes and
dihydroethidium. To enhance the signal, nanoparticles, such as
quantum dots and metallic nanoparticles, can be introduced. The
particles can be tagged with targeting molecules. Sorting
efficiency can be greater than 90%, while sperm viability rates are
greater than 30%, typically greater than 90%. Alternatively,
instead of electroporating the cells, the dye is introduced into
the cells by osmotic gradients. Cells are first incubated in
hypertonic conditions, and then transferred to hypotonic
conditions; the DNA-staining dye can be added to either, or both,
hypertonic and hypotonic solutions. After dying the cells, they are
ready to be sorted or further processed.
[0018] In a third aspect, the invention provides methods to
pre-select the sex of a mammalian offspring, where the sperm are
sorted according to the methods of the invention, and then
inseminating a female animal of the same species as the male animal
that provided the sperm. In a fourth aspect, instead of
inseminating a female animal, an egg from a female animal is
fertilized in vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A shows a schematic of an electroporation cell
[0020] FIG. 1B shows a schematic of a Resistance-Capacitator
circuit suitable for electroporation.
[0021] FIG. 2 outlines the steps for collecting, sorting and
freezing bovine sperm.
DETAILED DESCRIPTION
[0022] While the present invention is susceptible of embodiment in
many different forms, there are shown in the drawings and described
herein in detail specific embodiments thereof, with the
understanding that the present disclosure is to be considered as an
exemplification of the principles of the invention and is not
intended to limit the invention to the specific embodiments
illustrated.
[0023] The present invention solves the problem of breaching the
cell membrane barrier to introduce cellular stains and
nanoparticles without killing the cells. Using the methods of the
invention, populations of millions of living cells can be
simultaneously stained and are appropriate for further applications
that require living healthy cells because once applied, staining is
immediate.
[0024] In some embodiments of the invention, electroporation or
osmotic gradients are used to permeabilize cell membranes.
Electroporation passes an alternating or direct current (AC or DC)
electric field over the cells. The electric shock blasts holes into
the cell membrane. Under controlled conditions, the size of the
holes are big enough to allow introduction of the stain and/or
nanoparticles, but small enough to prevent excessive cytosol
leakage and irreparable cell damage that results in cell death.
Alternatively, osmotic pressure gradients are used to partially
dehydrate the cells. Cells are incubated in a hypertonic solution
and then transferred to a hypotonic solution; in either, or both
solutions, the dye is added. As the cells reach osmotic equilibrium
with the solution, water flows into the cell, drawing in the dye
across the cell membrane.
[0025] The methods of the invention also provide the unexpected
result of hastening the diffusion of membrane permeant dyes into
cells.
[0026] In addition to introducing stains and dyes into living
cells, the methods of the invention also allow the introduction of
nanoparticles that can be used as detectable entities in and of
themselves (e.g., quantum dots) or to amplify a signal, whether
innate to a target molecule or introduced. For example, metallic
nanoparticles create surface-enhanced resonances, amplifying the
natural fluorescence, auto-fluorescence, or fluorescently stained
molecules by orders of magnitude. Using metallic nanoparticles
therefore act as molecular mirrors, deflecting and augmenting
available light signals to which they are in close proximity. The
nanoparticles prevent energy loss of the stimulating radiation to
other modes, like phonons, and ensure that the energy is channeled
into emitted light. Because the natural fluorescence intensity of
some target molecules, such as DNA, is normally very low,
amplification the available signal reduces reliance on dyes or
stains which can interfere with normal functioning of the target
molecule. For example, many DNA-specific dyes intercalate between
the bases; this intercalation can, in mitotically or meiotically
active cells, introduce mutations into the genetic code.
[0027] Since the methods of the invention allow for fast live-cell
staining, other procedural parameters can be optimized to enhance
cell viability. For example, the time during which the cells are
mixed with the dye can be reduced or even eliminated, conserving
cellular resources. The temperatures at which the cells are
manipulated and held can also be reduced, effectuating slower
cellular metabolism that again conserves cellular resources.
[0028] The methods are especially appropriate for sorting sperm by
gender, in which quick staining of the sperm avoids the problems of
reduced viability because of prolonged incubation times.
DEFINITIONS
[0029] Cell-membrane-rupturing-force means force that is sufficient
to disrupt a cell membrane such that a cell-impermeant molecule is
able to cross the membrane. In the case of cell membrane-permeant
molecules, a disrupted membrane permits faster diffusion of the
molecule into the cell.
[0030] Comparatively high cell viability rate means a rate wherein
at least 5% of the total cell population (e.g., a population of
sperm) are alive. The rate can be determined by typical viability
tests, including exclusion of membrane impermeant dyes (e.g.,
trypan blue), or for monitoring for a specific cellular activity,
such as sperm locomotion.
[0031] DNA-staining dye means a detectable substance that interacts
with a polynucleotide such that when examined under appropriate
conditions, the polynucleotide is optically detected. While most
DNA-staining dyes interact directly with polynucleotides (such as
Hoechst stains), DNA-staining dyes also encompass those substances
that interact with molecules that interact with polynucleotides,
such as those that bind DNA-binding proteins, such as transcription
factors and histones. In some instances, DNA-staining dye molecules
consist of more than one molecule, such as an antibody tagged with
a detectable substance, the antibody specifically binding, for
example, a DNA-binding protein.
[0032] Electroporation means a phenomenon in which the membrane of
a cell, exposed to short, high intensity electric field pulses, is
temporarily destabilized in specific regions of the cell. During
the destabilization period, the cell membrane is highly permeable
to exogenous molecules present in the surrounding media.
Electroporation is one method of providing a cell
membrane-rupturing force.
[0033] Hypertonic condition means a condition in which the
concentration of electrolyte is above that found in cells in the
same solution. In this situation, osmotic pressure leads to the
migration of water from the cells to the surrounding solution in an
attempt to equalize the electrolyte concentration inside and
outside the cell.
[0034] Hypotonic condition means a condition in which the
concentration of electrolyte is below that found in cells in the
same solution. In this situation, osmotic pressure leads to the
migration of water into the cells in an attempt to equalize the
electrolyte concentration inside and outside the cell.
[0035] Nanometallic particle means a nano-scale structure
consisting of one or more metals, such as gold, silver, etc.
[0036] Permeating and related terms means to breach a cell
membrane. Permeating the cell membrane can be accomplished by
electroporation and osmotic stress, just as two examples.
[0037] Quantum dot means a nano-scale crystalline structure,
usually made from cadmium selenide, and absorbs white light and
then re-emits it a couple of nanoseconds later in a specific color.
The size of a quantum dot varies within the 10.sup.-9 m range, but
a quantum dot, regardless of size, is recognizable in that the
addition or subtraction of an electron represents a significant
change in the particle.
[0038] Targeting molecule means a molecule that has an affinity for
another molecule or group of molecules. Examples include
antibodies, streptavidin, avidin, biotin, etc.
[0039] Making and Using the Invention
[0040] Electroporation
[0041] When a short, high-voltage pulse surpasses the capacitance
of a cell membrane, transient--and reversible--disruption of a cell
membrane occurs (Gehl, 2003). This disruption allows for easier
diffusion of small molecules into the cell, as well as for
electrophoretically driving molecules through the destabilized
membrane (Gehl, 2003). Any electroporation (an electroporator) or
other cell membrane-rupturing-force device which parameters can be
manipulated as necessary by the user can be used in the methods of
the invention. Examples include the CUY21EDIT Square Wave
Electroporator and SONITRON2000 Sonoporator from Nepa Gene
(Ichikawa, Chiba; Japan); the EasyjecT Plus, EasyjecT Optima and
EasyjecT Prima (Flowgen; Nottingham, United Kingdom); Gene Pulser
Xcell System.TM. and MicroPulser Electroporator.TM. (BioRad
Laboratories; Hercules, Calif.). Typically, the resistance values
range from 2-10,000 ohms (.OMEGA.) depending primarily on the
electrical conductivity of the buffer. The capacitance varies from
0.1 millifarads (mF) to 1000 mF.
[0042] One embodiment of a suitable electroporation device is shown
in FIG. 1. Referring to FIG. 1A, the electroporation device
consists of two parallel glass slides 101 coated with 1,500-2000
angstroms (.ANG.) of indium tin oxide, which are separated by
fragments of number 0 glass cover slips 102, yielding a slide
separation of 100 millimeters (mm). Referring now to the circuit in
FIG. 1B, the sample cell 103 is connected to a resistor-capacitator
(RC) circuit by alligator clips. A direct current (DC) power supply
104 is used to charge a capacitor 105. When a switch is thrown, the
discharging capacitor generates a time-dependant and spatially
uniform electric field across the sample. An oscilloscope is used
to monitor the voltage across the sample cell as a function of
time. The RC circuit formed by the sample cell and capacitor allow
for a well-controlled electric field to be generated. The
resistance (R) of the circuit is left floating--that is, determined
by the geometry and content of the sample cell.
[0043] The temperature at which the cells are subjected to
electroporation varies with the cell type and the intended
application. For example, in the case of staining mammalian sperm
cells for sorting by gender, such as those from bovines, a
temperature of about -4.degree. C. to about 39.degree. C.;
preferably about 0.degree. C. to about 25.degree. C., more
preferably about 0.degree. C. to about 12.degree. C., and most
preferably about 0.degree. C., 1.degree. C., 2.degree. C.,
3.degree. C., 4.degree. C., 5.degree. C. and 6.degree. C.
[0044] Cells are suspended in an osmotically appropriate buffer for
electroporation, although the solution can be hyper- or hypotonic
to increase the efficiency of electroporation. For example, a 0.35
M sucrose solution yields good results for bovine sperm.
Appropriate biological buffers include Hank's Balanced Salt
Solution (HBSS), sodium phosphate-based buffers,
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris),
N-(2-hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or
HEPPS), glyclclycine,
N-2-hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES),
3-(N-morpholino)propane sulfonic acid (MOPS),
piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES), sodium
bicarbonate,
3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxypropanesulfonic
acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), and
tris(hydroxymethyl)aminomethane (Tris). Salt solutions that can be
used to produce osmotically appropriate conditions include
Alseverr's Solution, Dulbecco's Phosphate Buffered Saline (DPBS),
Earle's Balanced Salt Solution, Gey's Balanced Salt Solution
(GBSS), Puck's Saline A, Tyrode's Salt Solution, St. Thomas
Solution and University of Wisconsin Solution. In some instances, a
simple sucrose solution is sufficient; in others, a simple
buffer.
[0045] Because cells differ from organism to organism, of even the
same cell type, the parameters for electroporation may need to be
determined experimentally. An assay is provided to determine the
appropriate parameters for each cell type.
[0046] Assay to Determine Parameters for Electroporation
Introduction of Dyes and Nanoparticles
[0047] Cells are harvested according to established procedures,
preferably at 4.degree. C. or other metabolic-suspending
temperatures, washed, and re-suspended in an osmotically
appropriate buffer, preferably without additional divalent cations,
such as Ca.sup.2+, Mg.sup.2+ and Mn.sup.2+, at a concentration of
1-2.times.10.sup.7 cells per ml. The concentration of the cells can
be altered to accommodate the differences in cell size and other
variables. The desired dye and/or nanoparticles are added to the
suspension; the concentrations of which can be experimentally
determined. In the case of dyes wherein the binding sites are known
(e.g., intercalation between adenosine and threonine in DNA), an
estimate of the appropriate dye concentration can be calculated by
using an estimate of the amount of DNA in the sample. In some
cases, the cells, dye and/or nanoparticles are incubated in the
electroporation cell for a short period of time, e.g., 1-15
minutes, preferably 1-5 minutes, preferably less than 5 minutes,
more preferably less than 1 minute, and most preferably 30 seconds
at a metabolically-suspending temperature (e.g., 0-4.degree. C.).
In other instances, there is no pre-incubation and the cells are
electroporated immediately. In the case of nanoparticles,
incubation times are minimized to prevent any settling or non
random dispersion of the particles. Alternatively, the viscosity of
the buffer can be altered to maintain the suspension of particles,
such as the addition of a protein (e.g., bovine serum albumin) or
inert substance.
[0048] An electric pulse is applied; a starting voltage of 2.0 kV
represents a reasonable starting point, with the current set at a
maximum of 0.9 mA. Adjustable current and wattage dials are set at
bare minimum. In some cases, the cells, dye and/or nanoparticles
are incubated in the electroporation cell for a short period of
time after electroporation, e.g., 1-15 minutes, preferably 1-5
minutes, preferably less than 5 minutes, more preferably less than
1 minute, and most preferably 30 seconds at a
metabolically-suspending temperature (e.g., 0-4.degree. C.). In
other instances, no post-incubation step is necessary. For other
cell types, restoring physiological conditions is paramount to
preserve cell viability; for these cells, a recovery solution
(e.g., culture media) is added immediately. Cells are then
transferred for further processing (e.g., washing, collecting,
freezing) and observation. In most cases, a recovery media
containing divalent cations is preferable, such as provided in
appropriate growth media.
[0049] Table 1 presents just one example of an experimental design
(adapted from (Potter et al., 1984)); this example is not meant to
be limiting. One of skill in the art will know how to manipulate
these and other appropriate experimental parameters. TABLE-US-00001
TABLE 1 Example of experimental parameters for determining transfer
frequency by electroporation Cells Power supply settings (kV/mA)
Electrode Temperature (.degree. C.) A 1.2/300 Al 20 A 1.2/300 Al 20
Temperature and voltage effects A 1.2/300 Al 20 A 1.2/300 Al 0 A
1.2/300 Al 20 A 4.0/0.9 Al 20 A 4.0/capacitor Al 20 A 1.2/100 SS 20
A 1.2/100 ss 0 A 1.2/300 SS 0 A 2.0/0.9 ss 0 A 4.0/capacitor ss 0
Comparison of cell lines and species A 4.0/0.9 Al 0 B 4.0/0.9 Al 0
C 4.0/0.9 Al 0 D 4.0/0.9 Al 0
[0050] For bovine sperm, when introducing macromolecules such as
DNA, a pulse of 0.25 seconds at 25 .mu.F capacitance and 300 V is
sufficient in a 1.4 ml electroporation chamber where the electrodes
are 4 mm apart (Rieth et al., 2000). For smaller molecules, shorter
pulses (e.g., approximately 0.25 ms) at lower voltages (e.g.,
approximately 10 V) can be used (see Example 1).
[0051] Cells
[0052] Cells or tissue samples that are appropriate for the methods
of the invention are collected from a subject or a culture. The
subject can be a vertebrate, more preferably a mammal, such as a
bull, monkey, dog, cat, rabbit, pig, goat, sheep, horse, rat,
mouse, guinea pig, etc. Any technique to collect the desired cells
may be employed, including biopsy, surgery, scrape (inner cheek,
skin, etc.), induced ejaculation (for sperm) and blood withdrawal.
Any cultured cell type, whether ex vivo cultured cells from a
subject, or a cell line, such as Madin-Darby Canine Kidney (MDCK),
HeLa, CaCO-2, immunoglobulin-secreting hybridomas, etc. can also be
used in the methods of the invention.
[0053] Stains, Dyes and Other Visual Labels
[0054] To detect a molecule of interest, a label can be used. The
label can be coupled to a binding antibody or other interacting
polypeptide, or to one or more particles, such as a nanoparticle.
Suitable labels include fluorescent moieties, such as fluorescein
isothiocyanate; fluorescein dichlorotriazine and fluorinated
analogs of fluorescein; naphthofluorescein carboxylic acid and its
succinimidyl ester; carboxyrhodamine 6G; pyridyloxazole
derivatives; Cy2, 3 and 5; phycoerythrin; fluorescent species of
succinimidyl esters, carboxylic acids, isothiocyanates, sulfonyl
chlorides, and dansyl chlorides, including propionic acid
succinimidyl esters, and pentanoic acid succinimidyl esters;
succinimidyl esters of carboxytetramethylrhodamine; rhodamine Red-X
succinimidyl ester; Texas Red sulfonyl chloride; Texas Red-X
succinimidyl ester; Texas Red-X sodium tetrafluorophenol ester;
Red-X; Texas Red dyes; tetramethylrhodamine; lissamine rhodamine B;
tetramethylrhodamine; tetramethylrhodamine isothiocyanate;
naphthofluoresceins; coumarin derivatives; pyrenes; pyridyloxazole
derivatives; dapoxyl dyes; Cascade Blue and Yellow dyes; benzofuran
isothiocyanates; sodium tetrafluorophenols;
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. In some cases enzymatic
moieties can be appropriate, such as alkaline phosphatase or
horseradish peroxidase; and radioactive moieties, including
.sup.35[S] and .sup.135[I] labels. The choice of the label depends
on the application, the desired resolution and the desired
observation methods. For fluorescent labels, the fluorophore is
excited with the appropriate wavelength, and the sample observed
using a microscope, confocal microscope, or fluorescence-activate
cell sorting (FACS) machine. In the case of radioactive labeling,
the samples are contacted with autoradiography film and developed;
alternatively, autoradiography can also be accomplished using
ultrastructural approaches.
[0055] Dyes and stains that are specific for DNA (or preferentially
bind double stranded polynucleotides in contrast to single-stranded
polynucleotides) include Hoechst 33342
(2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole)
and Hoechst 33258
(2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole)
and others of the Hoechst series; SYTO 40, SYTO 11, 12, 13, 14, 15,
16, 20, 21, 22, 23, 24, 25 (green); SYTO 17, 59 (red), DAPI,
YOYO-1, propidium iodide, YO-PRO-3, TO-PRO-3, YOYO-3 and TOTO-3,
SYTOX Green, SYTOX, methyl green, acridine homodimer,
7-aminoactinomycin D, 9-amino-6-chloro-2-methoxyactridine. Tables
1, 2 and 3 list many of the available
polynucleotides-specific/chromosome specific stains currently
available (Tables 2-4 have been adapted from (Haugland, 2002)).
TABLE-US-00002 TABLE 2 Cell membrane-impermeant cyanine nucleic
acid stains Catalogue #.sup.1 Dye Name Ex/Em* Excitation
Source.dagger. SYTOX Dyes S11348 SYTOX Blue 445/470 Hg-arc lamp,
436 nm line S7020 SYTOX Green 504/523 Ar-ion laser, 488 nm line
S11368 SYTOX Orange 547/570 Nd: YAG laser, 532 nm line Cyanine
Dimers P3580 POPO-1 434/456 Hg-arc lamp, 436 nm line He--Cd laser,
442 nm line B3582 BOBO-1 462/481 Hg-arc lamp, 436 nm line He--Cd
laser, 442 nm line Y3601 YOYO-1 491/509 Ar-ion laser, 488 nm line
T3600 TOTO-1 514/533 Ar-ion laser, 514 nm line J11372 JOJO-1
529/545 Nd: YAG laser, 532 nm line P3584 POPO-3 534/570 Nd: YAG
laser, 532 nm line L11376 LOLO-1 565/579 Kr-ion laser, 568 nm line
B3586 BOBO-3 570/602 Hg-arc lamp, 578 nm line Y3606 YOYO-3 612/631
Orange He--Ne laser, 594 nm line T3604 TOTO-3 642/660 He--Ne laser,
633 nm line Kr-ion laser, 647 nm line 635 nm diode laser Cyanine
Monomers P3581 PO-PRO-1 435/455 Hg-arc lamp, 436 nm line He--Cd
laser, 442 nm line B3583 BO-PRO-1 462/481 Hg-arc lamp, 436 nm line
He--Cd laser, 442 nm line Y3603 YO-PRO-1 491/509 Ar-ion laser, 488
nm line T3602 TO-PRO-1 515/531 Ar-ion laser, 514 nm line J11373
JO-PRO-1 530/546 Nd: YAG laser, 532 nm line P3585 PO-PRO-3 539/567
Nd: YAG laser, 532 nm line He--Ne laser, 543 nm line L11377
LO-PRO-1 567/580 Kr-ion laser, 568 nm line B3587 BO-PRO-3 575/599
Hg-arc lamp, 578 nm line Y3607 YO-PRO-3 612/631 He--Ne laser, 594
nm line T3605 TO-PRO-3 642/661 He--Ne laser, 633 nm line Kr-ion
laser, 647 nm line T7596 TO-PRO-5 747/770 Laser diodes
.sup.1According to (Haugland, 2002), catalogue numbers are specific
to Molecular Probes, Inc. *Wavelengths of excitation (Ex) and
emission (Em) maxima, in nm. .dagger.Nearest major emission line of
some common light sources.
[0056] TABLE-US-00003 TABLE 3 Cell-permeant cyanine nucleic acid
stains Catalogue #.sup.1 Dye Name* Ex/Em.dagger. Blue-fluorescent
SYTO dyes S11351 SYTO 40 blue-fluorescent nucleic acid stain
419/445 S11352 SYTO 41 blue-fluorescent nucleic acid stain 426/455
S11353 SYTO 42 blue-fluorescent nucleic acid stain 430/460 S11354
SYTO 43 blue-fluorescent nucleic acid stain 437/464 S11355 SYTO 44
blue-fluorescent nucleic acid stain 445/472 S11356 SYTO 45
blue-fluorescent nucleic acid stain 452/484 Green-fluorescent SYTO
Dyes S34854 SYTO 9 green-fluorescent nucleic acid stain 483/503
S32704 SYTO 10 green-fluorescent nucleic acid stain 484/505 S34855
SYTO BC green-fluorescent nucleic acid stain 485/500 S7575 SYTO 13
green-fluorescent nucleic acid stain 488/509 S7578 SYTO 16
green-fluorescent nucleic acid stain 488/518 S7559 SYTO 24
green-fluorescent nucleic acid stain 490/515 S7556 SYTO 21
green-fluorescent nucleic acid stain 494/517 S32706 SYTO 27
green-fluorescent nucleic acid stain 495/537 S32705 SYTO 26
green-fluorescent nucleic acid stain 497/534 S7558 SYTO 23
green-fluorescent nucleic acid stain 499/520 S7574 SYTO 12
green-fluorescent nucleic acid stain 500/522 S7573 SYTO 11
green-fluorescent nucleic acid stain 508/527 S7555 SYTO 20
green-fluorescent nucleic acid stain 512/530 S7557 SYTO 22
green-fluorescent nucleic acid stain 515/535 S7577 SYTO 15
green-fluorescent nucleic acid stain 516/546 S7576 SYTO 14
green-fluorescent nucleic acid stain 517/549 S7560 SYTO 25
green-fluorescent nucleic acid stain 521/556 Orange-fluorescent
SYTO dyes 532707 SYTO 86 orange-fluorescent nucleic acid stain
528/556 S11362 SYTO 81 orange-fluorescent nucleic acid stain
530/544 S11361 SYTO 80 orange-fluorescent nucleic acid stain
531/545 S11363 SYTO 82 orange-fluorescent nucleic acid stain
541/560 S11364 SYTO 83 orange-fluorescent nucleic acid stain
543/559 S11365 SYTO 84 orange-fluorescent nucleic acid stain
567/582 S11366 SYTO 85 orange-fluorescent nucleic acid stain
567/583 Red-fluorescent SYTO dyes S11346 SYTO 64 red-fluorescent
nucleic acid stain 598/620 S11343 SYTO 61 red-fluorescent nucleic
acid stain 620/647 S7579 SYTO 17 red-fluorescent nucleic acid stain
621/634 S11341 SYTO 59 red-fluorescent nucleic acid stain 622/645
S11344 SYTO 62 red-fluorescent nucleic acid stain 649/680 S11342
SYTO 60 red-fluorescent nucleic acid stain 652/678 S11345 SYTO 63
red-fluorescent nucleic acid stain 654/675 .sup.1According to
(Haugland, 2002), catalogue numbers are specific to Molecular
Probes, Inc. .dagger.Wavelengths of excitation (Ex) and emission
(Em) maxima, in nm.
[0057] TABLE-US-00004 TABLE 4 Properties of classic nucleic acid
stains Fluorescence Catalogue #.sup.1 Dye Name Ex/Em* Emission
Color Applications.dagger. A666 Acridine homodimer 431/498 Green
Impermeant AT-selective High-affinity DNA binding A1310 7-AAD
(7-amino- 546/647 Red Weakly permeant actinomycin D) GC-selective
Flow cytometry Chromosome banding A1324 ACMA 419/483 Blue
AT-selective Alternative to quinacrine for chromosome Q banding
D1306, D3571, DAPI 358/461 Blue Semi-permeant D21490 AT-selective
Cell-cycle studies Chromosome and nuclei counterstain Chromosome
banding D1168, D11347, Dihydroethidium 518/605 Red.sctn. Permeant
D23107 Blue fluorescent until oxidized to ethidium E1305,
E3565.dagger-dbl. Ethidium bromide 518/605 Red Impermeant dsDNA
intercalator Dead-cell stain Chromosome counterstain Flow cytometry
Argon-ion laser excitable E1169 Ethidium homodimer-1 528/617 Red
Impermeant (EthD-1) High-affinity DNA labeling Dead-cell stain
Argon-ion and green He--Ne laser excitable E3599 Ethidium
homodimer-2 535/624 Red Impermeant (EthD-2) Very high-affinity DNA
labeling Electrophoresis prestain E1374 Ethidium monoazide 464/625
Red Impermeant (unbound)** Photocrosslinkable H1398,
H3569.dagger-dbl., Hoechst 33258 (bis- 352/461 Blue Permeant H21491
benzimide) AT-selective Minor groove-binding dsDNA-selective
binding Chromosome and nuclear counterstain H1399,
H3570.dagger-dbl., Hoechst 33342 350/461 Blue Permeant H21492
AT-selective Minor groove-binding dsDNA-selective binding
Chromosome and nuclear counterstain H21486 Hoechst 34580 392/498
Blue Permeant AT-selective Minor groove-binding dsDNA-selective
binding Chromosome and nuclear counterstain H22845
Hydroxystilbamidine 385/emission varies Varies AT-selective with
nucleic acid Spectra dependent on secondary structure and sequence
RNA/DNA discrimination L7595 LDS 751 543/712 (DNA) Red/infrared
Permeant 590/607 (RNA) High Stokes shift Long-wavelength spectra
Flow cytometry N21485 Nuclear yellow 355/495 Yellow Impermeant
Nuclear counterstain P1304MP, Propidium iodide (PI) 530/625 Red
Impermeant P3566.dagger-dbl., P21493 Dead-cell stain Chromosome and
nuclear counterstain .sup.1According to (Haugland, 2002), catalogue
numbers are specific to Molecular Probes, Inc. *Excitation (Ex) and
emission (Em) maxima in nm. .dagger.Indication of dyes as
"permeant" or "impermeant" are for the most common applications;
permeability to cell membranes may vary considerably with the cell
type, dye concentrations and other staining conditions. .sctn.After
oxidation to ethidium. **Prior to photolysis; after photolysis the
spectra of the dye/DNA complexes are similar to those of ethidium
bromide-DNA complexes.
[0058] In some cases, such as DNA and certain polypeptides, a
physical characteristic of that molecule can be used, such as the
innate autofluorescence in DNA. In such cases, signal intensity can
be modulated by the introduction of nanoparticles (see Modulating
fluorescence signals with nanoparticles, below).
[0059] Introducing Dye via Osmolarity/Osmolality Modulation
[0060] The modulation of the concentration of solutes can create an
environment that is either hypertonic or hypotonic to cells. By
suspending the cells in a hypertonic solution, cells become
partially dehydrated. After a short period, they are then
transferred to a hypotonic solution. Either, or both solutions can
include the dye of interest, but should be present to be available
to the cells to enter the cells. Preferably, the dye is present in
at least the hypotonic solution. As the cells reach osmotic
equilibrium with the solution, water flows into the cell, drawing
in the dye across the cell membrane.
[0061] Osmolality can be varied by either adding appropriate salts
or other solutes that are compatible with the cells of interest
(e.g., KCl, NaCl, MgCl.sub.2, MnCl.sub.2, CaCl.sub.2, sucrose,
glucose, etc.), or by diluting the solution with water or buffer.
After collection, cells are transferred to a hypertonic solution
for about 0 to about 15 minutes, preferably, about 1 to about 10
minutes, more preferably about 3 to about 7 minutes, and most
preferably about 5 minutes. The temperature of the solution is
about -4.degree. C. to about 39.degree. C., preferably about
0.degree. C. to about 25.degree. C., more preferably 0.degree. C.
to about 12.degree. C., and most preferably, about 4.degree. C. The
cells are then transferred to a hypotonic solution or the solution
in which the cells are in is diluted with buffer to create
hypotonic conditions. The temperature of the added solution is bout
-40 C. to about 39.degree. C., preferably about 0.degree. C. to
about 25.degree. C., more preferably 0.degree. C. to about
12.degree. C., and most preferably, about 4.degree. C.
[0062] Osmolality conditions vary somewhat by cell type. However,
for bovine sperm, hypertonic conditions are created at
approximately greater than 250 mOsm, whereas hypotonic conditions
are created at approximately less than 250 mOsm (Liu and Foote,
1998). Preferred hypertonic osmalities include 100 mOsm to 249
mOsm; most preferably greater than 150 mOsm, but less than 250
mOsm. Hypotonic osmalities include 251 mOsm to 1537 mOsm;
preferably 500 mOsm to 963 mOsm; and most preferably greater than
250 mOsm but less than 732 mOsm. The dye can be any of those listed
in Tables 2-4 or other appropriate dye.
[0063] Modulating Fluorescence Signals with Nanoparticles (Quantum
Dots and Metallic Nanoparticles)
[0064] Organic and biomolecular fluorophores generally exhibit only
moderate Stokes shifts between their excitation and emission
spectra, have relatively broad emission spectra, and photobleach
when monitored over extended periods of time. A promising
alternative to conventional fluorophores is quantum dots (QDs)
(Doty et al., 2004).
[0065] In one embodiment, the core of a QD consists of a
semiconductor nanocrystal, such as CdSe, surrounded by a
passivation shell, such as ZnS. Upon absorption of a photon, an
electron-hole pair is generated, the recombination of which in
.about.10-20 ns leads to the emission of a less-energetic photon.
This energy, and therefore the wavelength, is dependent on the size
of the core (smaller->lower wavelength), which can be varied
almost at will by controlled-synthesis conditions (Lidke and
Arndt-Jovin, 2004). The surface is coated with a polymer that
protects the QD from water and allows for chemical coupling to
molecules.
[0066] The excitation spectra of QDs are a continuum, rising into
the ultraviolet, and the emission spectra are narrow and slightly
red-shifted to the band-gap absorption. Thus QDs with different
emissions can be excited with a single excitation (Smith and Nie,
2004). The large extinction coefficient and the relatively high
quantum yield of QDs, as well as their extraordinary
photostability, permit the use of a low sample irradiance and
prolonged imaging with a detection sensitivity extending down to
the single-QD level.
[0067] QDs are commercially available (e.g., Quantum Dot Corp.;
Hayward, Calif. and Evident Technologies; Troy, N.Y.) with a
variety of conjugated or reactive surfaces, e.g., amino, carboxyl,
streptavidin, protein A, biotin, and immunoglobulins. QDs are
non-toxic to most cells. For example, tissue culture cells loaded
with QDs survive for weeks without diminished growth or division,
and the QDs persisted the entire time (Doty et al., 2004). In live
animal studies, mice lived normal lives with QDs for months without
obvious deleterious effects (Lidke and Arndt-Jovin, 2004). QDs can
be introduced into sexual reproductive cells without harm. For
example, Xenopus embryos injected with QDs did not alter the
subsequent phenotype; the QDs were viewable throughout development
(Smith and Nie, 2004).
[0068] QDs can be targeted to specific areas of the cell, such as
the nucleus, by coating them with appropriate molecules, such as
DNA-binding molecules (oligonucleotides, DNA-binding proteins, such
as histones, transcription factors, polymerases and other molecules
of the chromatin, DNA-binding dyes, such as those listed in Tables
2-4, or other small molecules, such as other base intercalators).
The particles are suspended with the cells prior to
electroporation.
[0069] Similarly, metallic nano-particles can be used to enhance
any fluorescent signal, such as those made of gold and silver. They
can likewise be tagged with targeting molecules such that they are
in close proximity of the stained DNA.
[0070] Dectecting Chromosomal Differences with Nanotransistors and
Photo-Activatable Fluorophores
[0071] The ability to incorporate an indicator that has a strongly
non-linear response to DNA amount facilitates measuring DNA
content. Ideally, this indicator has very little or no fluorescence
for the amount of DNA associated with one chromosome, and large
amounts of fluorescence for the amount of DNA associated with
another chromosome, although in practice approximations to this
non-linear response curve are extremely useful. There are several
mechanisms that exist for generating such non-linear fluorescence.
Photo-activated fluorophores are one such mechanism. Incorporating
a photo-activated fluorophore which has a non-linear response to
the usual fluorescence emitted by a DNA stain provides a flexible
combination of fluorophores whose properties may be tuned to
achieve the desired non-linearity (Hogan, 2005). Such
photo-activated fluorophores would not necessarily have to be
incorporated into the nucleus of the sperm as they may be tuned to
respond to fluorescence from the sperm as a whole. By incorporating
molecular or nano-transistors into the medium or the sperm, they
act as non-linear amplifiers for fluorescence radiated from DNA,
either innate autofluorescence or that generated from a stain. Such
nano-transistors have countless embodiments because they can be
based on biological proteins, or based on quantum dot transistors.
There is a significant advantage in that the light that is measured
is provided by a strong "pumping" source as opposed to the weak
gating source that is usually associated with natural fluorescence
of DNA or staining, like Hoescht stains and others listed in Tables
2-4.
[0072] Metallic Nanoparticles and Other Modifiers of Fluorophore
Free-Space Spectral Properties
[0073] Nearby conducing metallic particles, colloids or surfaces
can modify free-space spectral conditions of fluorophores such that
the incident electric field "felt" by the fluorophore is increased
(or decreased), and the rate of radiavity decay can also be
modulated (Asian et al., 2004). The radiavity decay rate is that at
which a fluorophore emits photons. Because the metallic
nanoparticles need to be in close proximity to the fluorescent
molecule (approximately about 5 nm), particles can be tagged with
fluorescent molecules; or, in the case of polynucleotides (which
have a low level of auto fluorescence at 260 nm and 280 nm), tagged
with molecules that bind the polynucleotides, such as
oligonucleotides, small molecules, or polynucleotide specific
binding polypeptides. The particles are suspended with the cells
prior to electroporation.
[0074] Flow Cytometry/Fluorescence-Activated Cell Sorting
(FACS)
[0075] Methods of performing flow cytometry are well known (Lidke
and Arndt-Jovin, 2004). Flow cytometry (measurement of cells as
they flow by a detector) has been available for analysis and
sorting a variety of cell types in fluid suspension since the late
1970s. Flow cytometers use focused laser light to illuminate cells
as they pass the laser beam, one at a time, in a fine fluid stream.
Light scattered by the cells and light emitted by fluorescent dyes
attached or loaded in the cells are analyzed by detectors. Cells
can be distinguished and selected on the basis of size and shape,
as well as by the presence of different molecules inside and on the
surface of the cells.
[0076] FIG. 2 outlines the flow that can be applied to
gender-sorting of sperm. The sperm are collected from the donor 201
and subjected to extension 202 and then cooled slowly to 6.degree.
C. Once cooled, the sperm can be subjected to staining DNA by any
technique, but preferably the novel techniques of the present
invention, using electroporation or osmolality and/or nanoparticles
of various compositions. The stained cells are introduced into a
cell sorting device 204 and separated based on gender difference,
usually by the sex chromosomes X and Y. The sorted cells are
collected, and slowly cooled to 4.degree. C. 205 before being
subjected to a final extension 206. The cells are loaded into
cryogenic-compatible straws 207, the cell allowed to settle 208,
and then frozen 209.
[0077] In the methods of the invention, because of the advantages
of staining cells at 4.degree. C. or even cooler, the cells may be
cooled to greater than 6.degree. C. as shown in step 203. Sorting
itself 204 can also take place at cooler temperatures, thus
preserving cell integrity and cell viability. In many bovine sperm
separation protocols, eggs, egg yolks or other sperm-supporting
substances are added to the collected sperm to improve viability;
however, because the present invention allows for staining and
sorting of the cells at cool temperatures--those in which the cells
are metabolically inactive, or nearly so ("metabolically
suspended")--such a step (and the potential of introducing a
confounding variable in sorting, being obliged to pre-filter before
sorting, and potentially contaminating the sample with microbes)
can be eliminated, or the quantity of added egg or other
sperm-supporting substances can be reduced to facilitate sorting
and other processing.
[0078] Eliminating Dead Cells
[0079] After electroporation or osmotic introduction of dyes and/or
nanoparticles, a portion of the cell population will usually be
nonviable. To increase the quality of the output of the cell
populations at the end of processing the cells, dead cells can be
removed from the live cells.
[0080] In the case of using fluorescent dyes, such as those that
bind DNA listed in Tables 2-4, dead cells and successfully stained,
viable cells, both fluoresce. This is because dead cells have
compromised cell membranes. To eliminate dead cells from the
population, one approach is to add a counter-stain that diminishes
the signal. For example, in sperm, membrane-impermeant red food
coloring is mixed with the cells. By first dyeing the sperm with an
impermeable dye, the Hoechst dye is not able to bind to the DNA
since the impermeable dye is already bound. Therefore, the
fluorescence of the dead sperm should be either eliminated or a
different color, depending on the impermeable DNA dye used. Other
classic tests include Trypan blue exclusion, where only non-viable
cells allow entry of the dye, diminishing fluorescent signals. In
this case, the dye is added after electroporation or osmotic shock,
but before sorting.
EXAMPLES
[0081] The following example is for illustrative purposes only and
should not be interpreted as limitations of the claimed invention.
There are a variety of alternative techniques and procedures
available to those of skill in the art which would similarly permit
one to successfully perform the intended invention.
Example 1
In vivo Staining of Sperm Cells with a DNA-Specific Dye
[0082] Methods and Materials
[0083] Electroporation unit A sample cell was formed by two
parallel glass slides coated with 1,500-2000 angstroms (.ANG.) of
indium tin oxide (ITO). The slides were separated by fragments of
number zero glass cover slips, yielding a slide separation of 100
millimeters (mm).
[0084] The sample cell was connected to a resistor-capacitator (RC)
circuit by alligator clips. A direct current (DC) power supply was
used to charge a capacitor. When a switch was thrown, the
discharging capacitor generated a time-dependant and spatially
uniform electric field across the sample. An oscilloscope was used
to monitor the voltage across the sample cell as a function of
time.
[0085] The RC circuit formed by the sample cell and capacitor
allowed for a well-controlled electric field to be generated. The
resistance (R) of the circuit was left floating--that is,
determined by the geometry and content of the sample cell. Typical
R values ranged from 2-10,000 watts (W) depending primarily on the
electrical conductivity of the buffer. The capacitance was varied
from 0.1 millifarads (mF) to 1000 mF.
[0086] Cells Bovine sperm that had been previously frozen were
thawed for 60 seconds in a 96.degree. F. water bath. Sperm were
then centrifuged at 2,000 rotations per minute (rpm) for two
minutes and the supernatant decanted. Sperm were then re-suspended
with 0.35 M sucrose to partially dehydrate the sperm. The sperm
solution was then incubated at 96.degree. F. for 15 minutes and
then transferred to the sample cell of the electroporation
unit.
[0087] Electroporation A voltage was applied to the capacitor
circuit, and then the power supply was disconnected. A switch was
then thrown and the capacitor discharged across the sample cell.
These steps were carried out within 15 seconds of transferring the
sperm to the sample cell to retain a random orientation of sperm
with respect to the electric field. Electroporation was carried out
on sperm in isotonic (0.25 M sucrose) and hypertonic (0.35 M
sucrose) solutions. In each case, 10 V was applied to the capacitor
and a time constant of 0.26 milliseconds (ms) measured.
[0088] A solution of 0.1 M sucrose and the DNA-specific dye,
propidium iodide, was then injected into the sample cell.
Fluorescence microscopy is used to image the sperm.
[0089] Results
[0090] Since propidium iodide dye does not breach the cell membrane
barrier, only cells that have had their membranes compromised, such
as by electroporation, allow entry of the dye, which then binds to
any DNA in the cell (the nucleus and mitochondria), and, when
excited with the appropriate wavelength of light, fluoresces. To
distinguish fluorescent dead cells from fluorescent live cells,
sperm motility was assessed.
[0091] In each case (isotonic and hypertonic/hypotonic solution),
motile and fluorescent sperm were observed. However, the number of
these sperm was enhanced by approximately five-ten fold in the
hypertonic/hypotonic solution. Control samples unexposed to
electric fields did not yield motile and fluorescent sperm.
[0092] Motility was examined in the hypertonic/hypotonic solution
and compared to a control sample unexposed to an electric field.
The electroporated sample yielded a loss of 70% of motile sperm
compared to the control. In addition, the electroporated sample
showed a 68% increase in the number of damaged and non-motile
sperm. All of the observed motile sperm in the hypertonic/hypotonic
solution exposed to the electric field displayed fluorescence.
[0093] Larger field strengths, multiple pulses, alternating current
(AC) fields, buffers with larger osmotic pressures, and longer time
constants led to complete loss of sperm motility. Weaker field
strengths and shorter time constants did not yield fluorescent
motile sperm.
[0094] A temporary spatially uniform electric field allows
non-permeant membrane dyes to cross bovine sperm cell membranes. A
70% loss in motility was associated with this process--but
manipulating procedure parameters can reduce the death toll. This
technique also allows for the introduction of nanoparticles into
sperm and other membrane bound-cells.
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