|Molecular Vision 2004;
Received 15 September 2004 | Accepted 17 November 2004 | Published 17 November 2004
Targeted gene expression in the chicken eye by in ovo electroporation
Catherine Ellen Krull,3
Lixing Wang Reneker1,2
Departments of 1Ophthalmology and 2Biochemistry, University of Missouri, Columbia, MO; 3Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI
Correspondence to: Lixing W. Reneker, EC214 Mason Eye Institute, One
Hospital Drive, Columbia, MO, 65212; Phone: (573) 884-0350; FAX: (573)
884-4100; email: firstname.lastname@example.org
Dr. Chen is now at the Abramson Family Cancer Research Institute, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA, 19104
Purpose: The chicken embryo lens is a classical model system for developmental and cell biology studies. To understand the molecular mechanisms that underlie the morphological changes that occur during lens development, it is important to develop an effective gene transfer method that permits the analysis of gene functions in vivo. In ovo electroporation has been successfully used for introducing DNA into neural and mesenchymal tissues of chicken embryos. In this study, we explored the possibility of using this technique to manipulate gene expression in lens epithelial and fiber cells, as well as in other cells of the chicken eye.
Methods: Two DNA constructs were used in this study. pCAX contains a chicken β-actin promoter fused to the CMV IE enhancer to drive enhanced green fluorescent protein (EGFP) expression. pMES-cNf2 uses the same chimeric promoter to drive the expression of the chicken neurofibromatosis 2 (cNf2) and EGFP proteins in the same cell. Plasmid DNA was injected into the lumen of the lens vesicle in chicken embryos at stage 15. For corneal epithelial and retinal cell electroporation, DNA was placed near the surface ectoderm in the eye region or injected into the vitreous cavity, respectively. Electroporation was performed with one electrode above the eye and the other underneath the head of the embryo. Chicken embryos were harvested at different time points for EGFP expression analysis by immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) incorporation assays were used to evaluate the effects of cNf2 on lens epithelial cell proliferation.
Results: A strong EGFP signal can be detected in lens cells 4 h after electroporation. The transfected cells maintain high levels of EGFP expression for at least 5 days. Overexpressing cNf2 in lens epithelial cells significantly inhibits cell proliferation. Ectopic expression of EGFP in corneal epithelial and retinal cells was also achieved by in ovo electroporation.
Conclusions: We have demonstrated that exogenous DNA can be effectively introduced into lens, corneal and retinal cells in the living embryo by in ovo electroporation. In comparison to viral infection and transgenic mouse approaches, in ovo electroporation offers an easier and quicker way to manipulate gene expression during embryonic development. This technique will be a useful tool for exploring the molecular mechanisms of lens and eye development.
The embryonic lens is an attractive model system to study the molecular mechanisms that regulate cell proliferation and differentiation [1-4]. The chicken lens has a long history serving as an in vivo model for such studies because of its easy accessibility for surgical manipulation [5-8]. For example, in the classical lens inversion experiment of Coulombre and Coulombre , the chicken embryo lens was surgically rotated 180° so that the epithelial cells faced the vitreous body and neural retina. As a result, the former lens epithelial cells elongated and differentiated into fiber cells in a few days. This experiment provided evidence that the inductive signals for lens fiber cell differentiation were present in the vitreous and likely originated from the retina. In the past a few decades, although much effort has been focused on identifying the inducers for lens epithelial to fiber cell differentiation [8,10-12], the endogenous factors for lens fiber cell differentiation have not identified in either chicken or mammalian eyes.
A powerful approach to reveal the molecular mechanisms of development is to manipulate gene expression and examine the consequences in vivo. With a lens specific promoter to drive transgene expression, the mouse lens has been widely used for gene function analysis [13-16]. For example, different members of the FGF family have been shown to induce premature differentiation of the anterior lens epithelial cells in transgenic mice [17,18]. Blocking FGF receptor (FGFR) activity in the mouse lens is carried out by expressing either a dominant negative form of FGFR1 [19-21] or a secreted form of FGFR3 . This blocking inhibited normal lens fiber cell differentiation and resulted in smaller lenses in transgenic mice. These experiments suggest that, at least in the mouse, one or more members of the FGF family may play a critical role in fiber cell differentiation. Such experiments would be difficult to perform in the chicken embryo lens because of its limited accessibility to genetic manipulation. Therefore, despite the fact that much of our knowledge on lens development is based on the studies using the chicken embryo lens, it still remains unclear whether the signaling mechanism for fiber cell differentiation is evolutionally conserved between mammals and birds.
In recent years, in ovo electroporation has become a powerful tool to examine gene function during chicken embryonic development [23,24]. The basic steps of this technique  include (1) preparing a DNA construct to express the gene of interest, (2) microinjecting the DNA into a luminal space surrounded by the cells to be targeted, and (3) positioning the electrodes and applying current. The electric field pulses generated during electroporation cause a transient and reversible breakdown of the plasma membrane, and drive the negatively charged DNA to enter the cells that lie adjacent to the positive electrode (anode). In ovo electroporation has been used successfully to introduce genes into neural and mesenchymal tissues [26-28]. This method has been the most effective for ectopic or over-expression of gene products, an approach somewhat similar to the use of a tissue specific promoter to express particular genes in the transgenic mouse. Recently, in ovo electroporation has also been used for loss of function experiments [26,29-32]. In these studies, genes encode dominant negative proteins, or inhibitor RNAs such as RNAi or morpholino were introduced in the cells to knockdown endogenous gene products.
Surprisingly, in ovo electroporation has not been used as widely in chicken eyes as in other systems, although the first published study using electroporation was done in a chicken retinal explant . In theory, the structure of the optic and lens vesicles offers a great advantage for in ovo electroporation, since the DNA can be deposited into these luminal spaces. A few investigators have used this method to transfect the surface ectodermal (i.e., lens precursor) cells with transcription activators to study lens induction and specification [34-36]. However, it is not clear whether in ovo electroporation can be used to transfect more mature lens cells or other ocular cells. In this study, we demonstrate effective electroporation of cells in the lens vesicle and in the cornea and retina. Using in ovo electroporation, neurofibromatosis 2 (Nf2) cDNA was introduced into lens epithelial cells and shown to inhibit cell proliferation in chicken lens. This finding suggests that we can use this method to quickly identify and analyze gene function in lens or eye development.
The plasmid constructs used in this study were described previously [25,37]. The control plasmid pCAX, shown in Figure 1A, contains the coding sequences for enhanced green fluorescent protein (EGFP) driven by a chicken β-actin promoter fused to CMV IE enhancer. The experimental plasmid pMES-cNf2 (Figure 1B) consists of the chimeric CMV IE enhancer/β-actin promoter and the chicken Nf2 cDNA, followed by an internal ribosome entry site (IRES) and the EGFP coding sequences. After the plasmid is introduced into the cell by electroporation, a bicistronic mRNA will be generated, and Nf2 and EGFP proteins will be independently translated in the same cell. Plasmid DNA was prepared using the EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in PBS at a final concentration of 2 μg/μl.
Assembly of the electroporation apparatus
A BTX ECM 839 square Wave Electroporator with a foot pedal was used to generate the electric pulses during electroporation. Electrodes made of platinum wire at 0.01 inch in diameter (Newark Electronics, Chicago, IL) were insulated to about 5 mm from the tip, and the top 3 mm of the electrode was bent at a 135° angle. The electrodes were connected to the output wires of the electroporator.
The micropipette used for DNA injection was pulled from a capillary glass tube with 0.8-1.10 mm diameter and 100 mm length (A-M Systems, Carlsborg, WA) by a horizontal pipette puller (Sutter Instrument, Novato, CA). A few crystals of fast green were mixed with the DNA solution to monitor the location of DNA after injection. The micropipette was filled with fast green-positive DNA using a #805 Hamilton syringe and then placed in a needle holder connected to a micromanipulator (Narishige, East Meadow, NY). DNA injection was driven by a Picospritzer (Intracel, Royston, UK) connected to a nitrogen tank.
In ovo electroporation of plasmid DNA into ocular cells
Fertilized white Leghorn chicken eggs (Hy-Line International, Spencer, IA) could be stored at 15 °C for a few days if not used immediately for experiments. We calculated that most of the chicken embryos developed to stage 15 after incubating for about 56 h at 38 °C as evaluated by their morphological characteristics such as the number of somites (25 somites at stage 15) in the embryos. Embryos were prepared for DNA injection and electroporation as previously described [25,37].
Electroporation was performed on the right eye. The vitelline membrane overlying the right eye was carefully removed using a sharpened tungsten needle. To introduce plasmid DNA into the lens cells, a glass needle was slowly placed into the lens vesicle and used to deliver fast green-positive DNA solution (Figure 2A). After injection, approximately 2 ml of sterile Ringer's solution was applied on top of the embryo. To introduce DNA into the lens epithelial cells, the anode was placed in the Ringer's solution about 2 mm above the right eye and the cathode was inserted about 2 mm underneath the left eye in the space between the blastoderm and yolk (Figure 2B). For electroporating the lens fiber cells, the placement of the electrodes was reversed (Figure 2C). Five pulses at 20 V and 50 ms duration were applied. After removal of the electrodes, the window on the eggshell was sealed with Scotch magic tape and the embryos were returned to the incubator to develop to the appropriate stage.
To transfect prospective corneal epithelial cells, plasmid DNA was placed over the surface ectoderm at the eye region. For neural retinal cell transfections, plasmid DNA was injected into the vitreous cavity. Three pulses at 18 V and 50 ms duration were applied.
Immunohistochemistry of EGFP and αA-crystallin
The electroporated embryo in the egg was examined under a fluorescent stereomicroscope to detect the intrinsic fluorescence of EGFP. The EGFP expressing embryos were harvested at different time points after electroporation and rinsed with Ringer's solution. Membranes attached to the embryos were removed under a dissecting microscope. Embryos were fixed in 4% paraformaldehyde for 2 h and stored in PBS at 4 °C for further analysis.
To examine EGFP and αA-crystallin expression, 10 μm frozen sections were cut and incubated with a blocking solution consisting of 2% normal goat serum, 1% BSA, and 0.02% Tween-20 in PBS for 30 min. Tissue sections were incubated with primary antibodies against either EGFP (1:1000 dilution, Synaptic Systems, Goettingen, Germany) or αA-crystallin (1:100 dilution, a gift from Dr. David Beebe) for 2 h, washed with PBS, and then incubated with either FITC or rhodamine conjugated secondary antibody (1:500, Molecular Probes, Eugene, OR) for 1 h. Slides were mounted in Gel/Mount Media (Biomeda, Foster City, CA). Fluorescence was visualized by a Leica DMR microscope, and images were captured by a CCD camera and compiled using Adobe Photoshop 7.0. Although bright intrinsic fluorescence of EGFP can be readily detected in live embryos under fluorescent stereomicroscope, it is noteworthy that this EGFP signal sometimes diminishes in the pretreatment procedures used for detecting other proteins by immunohistochemistry. Therefore, we chose to use indirect immunohistochemical staining to co-localize EGFP with other antigens in this study.
5-bromo-2-deoxyuridine (BrdU) labeling and cell proliferation assay
Cell proliferation assays were performed following procedures described previously . In brief, 100 μl of 40 mM BrdU (Sigma, St. Louis, MO) solution was applied to the vitelline membrane of the electroporated embryos which were resealed and returned to the incubator at 37 °C for 30 min. Embryos were harvested, fixed with 4% paraformaldehyde in PBS for 1 h, and sectioned at 10 μm by a cryostat. Sections were labeled with anti-GFP antibodies followed by an anti-rabbit IgG-FITC secondary antibody to reveal the transfected cells. To detect BrdU positive cells, sections were treated with 2 N HCl at 37 °C for 30 min and then labeled with anti-BrdU antibody (1:200, Sigma) and an anti-mouse IgG-Cy5 secondary antibody. Cell nuclei were stained by propidium iodide (10 μg/ml, Molecular Probes) for 30 min at room temperature. Images were acquired by a BioRad confocal microscope and assembled using Photoshop 7.0. The cell proliferation rate was calculated based on the percent of BrdU-EGFP double positive cells in total EGFP positive cells. The numbers were evaluated using a student t-test.
Targeted expression of EGFP in lens epithelial and fiber cells by electroporation
To test for in ovo electroporation of lens cells, we used EGFP as a reporter gene. Plasmid DNA (pCAX in Figure 1A), at a concentration of 2 μg/μl, was microinjected into the lumen of the lens vesicle of the right eye of the chicken embryo at stage 15-16 (Figure 2A). To drive the negatively charged DNA into the lens epithelial cells, the positive electrode (anode) was placed in the Ringer's solution above the right eye and the negative electrode (cathode) in between the blastoderm and yolk underneath the left eye (Figure 2B). Different electroporation parameters were tested to achieve high efficiency with minimal tissue damage and cell death. We found that five pulses at 20 V and 50 ms duration was optimal for electroporation in lens cells. After electroporation, the live embryos in eggs were examined under a fluorescent stereomicroscope to detect the intrinsic fluorescence of EGFP. The earliest time we detected EGFP expression was 4 h post-electroporation. To confirm this observation, chicken embryos were harvested and lens sections were stained with anti-GFP antibody (Figure 3A, Figure 3E, and Figure 3I) and with anti-αA-crystallin antibody to localize the lens cells (Figure 3B, Figure 3F, and Figure 3J). EGFP immunofluorescence was localized exclusively in the anterior cells of the lens vesicle (Figure 3A). As the lens development proceeds, the anterior cells remain proliferative and form the lens epithelium, and the posterior cells elongate and differentiate into fiber cells to fill up the lumen (Figure 3E-I). At stage 21, about 24 h after electroporation, EGFP expression was still limited to the lens epithelial cells (Figure 3E and Figure 3H). After 48 h, EGFP expression was detected not only in the lens epithelial cells, but also in the cortical fiber cells (Figure 3I and Figure 3L). The EGFP expressing fiber cells are likely differentiated from the transfected lens epithelial cells close to the equatorial zone. Thus, we have demonstrated that in ovo electroporation can be used to effectively introduce DNA into the lens epithelium for EGFP expression.
For fiber cell specific expression of EGFP, the placement of the electrodes was reversed (Figure 2C). The same electroporation parameters were applied. EGFP expression in lens fiber cells can be detected at 4 h post-electroporation (data not shown). The electroporation efficiency was extremely high in some cases, as shown in Figure 4A, almost all the primary fiber cells express high levels of EGFP at 24 h post-electroporation. At 48 h after electroporation, the EGFP-transfected primary fiber cells have been displaced to the central zone (Figure 4E and Figure 4H). The peripheral of the lens is negative for EGFP, because the cells in this region are new fiber cells that differentiated from untransfected epithelial cells. Five days post-electroporation, EGFP was still detected in the primary fiber cells in the central zone. In general, the rate of successful fiber cell electroporation was typically over 50%, which was higher than that of lens epithelial cell electroporation (over 30%).
Ectopic expression of c-merlin/EGFP in lens epithelial cells inhibits cell proliferation
We chose the NF2 gene to test the applicability of using in ovo electroporation for in vivo gene function analysis. NF2 is an autosomal dominant inherited disorder caused by inactivating mutations in the NF2 gene . NF2 patients develop nervous system tumors, including schwannomas, meningiomas, and ependymomas. Additionally, about half of NF2 patients also develop juvenile onset posterior subcapsular cataracts. The gene product of NF2, merlin (or schwannomin), is thought to function as a tumor suppressor gene although the detailed mechanism is still under investigation [39,40]. When Nf2 is conditionally knocked out using the Cre-loxP system with a Schwann cell promoter, mice develop many of the characteristics of NF2, including defects in the lens, confirming the important role of merlin in lens development .
Previously, we identified the chicken homolog of Nf2 (cNf2) and examined the localization of chicken merlin (c-merlin) during embryonic development . We found that c-merlin is expressed in lens epithelial cells and at a much higher level in lens fiber cells (data not shown). To test whether c-merlin can act as a negative growth regulator as it does in other types of cells , we electroporated lens epithelial cells in chicken embryos at stage 15 with cNf2/EGFP (Figure 5A-D) using EGFP-transfected lens epithelial cells as a control (Figure 5E-H). To monitor any changes in cell proliferation, we labeled the embryos at stage 21 (about 24 h after electroporation) with BrdU for 30 min before harvesting. The lens sections were stained with anti-GFP and anti-BrdU antibodies to identify transfected cells and cells that were in S-phase of the cell cycle, respectively. Cell nuclei were revealed by propidium iodide staining to facilitate cell counts.
We calculated the cell proliferation index of the transfected lens epithelial cells, defined as the number of BrdU-EGFP double positive cells divided by the total number of EGFP positive cells. Because of the inevitable variations among each electroporation, due to individual difference in embryos, position of the electrodes, and volume of the DNA injected, it is crucial to interpret data on the basis of a sufficient number of similar experiments. Thus, we counted a total of 1,099 and 966 EGFP positive cells in 5 control and 5 experimental embryos, respectively (Figure 5I). We found that the BrdU labeling index was about 42% in EGFP expressing control cells and 27% in c-merlin/EGFP expressing cells, suggesting that c-merlin expression in the lens epithelial cells inhibits cell proliferation. At 24 h post-electroporation, the c-merlin expressing lens cells were not elongated and they appeared morphologically normal.
Targeted EGFP expression in corneal and retinal epithelial cells
We also tested whether in ovo electroporation can be used to introduce DNA into corneal and retinal cells. For corneal epithelial cells, EGFP control plasmid DNA was applied near the surface ectoderm at the eye region. The placement of the electrodes was the same as in lens fiber cell electroporation shown in Figure 2C. Three electric pulses at 18 V and 50 ms duration were applied. After 24 h of incubation, EGFP signal was expressed in most of the corneal epithelial cells (Figure 6A-C).
To target DNA into the neural retina, plasmid DNA was microinjected into the vitreous cavity. Using the same parameters and electrode placement as for corneal epithelial cell electroporation, we observed strong EGFP expression in the neural retina after 24 h of incubation (Figure 6D-F). Therefore, in ovo electroporation can also be used for ectopic expression of exogenous genes in corneal and retinal cells.
The chicken embryonic eye is a classical model system to investigate questions in cell and developmental biology. To understand the molecular mechanisms underlying developmental processes, it is crucial to have an effective gene transfer method that allows us to study gene function and regulation in vivo. For the past few years, in ovo electroporation has been a well established method to introduce DNA into neural and mesenchymal tissues in chicken embryos [23,24]. In this study, we have demonstrated that we can use this method to ectopically express exogenous DNA at specific sites of the chicken eye, particularly in the lens epithelial and fiber cells. Plasmid DNA was microinjected into the lumen of the lens vesicle in chicken embryos at stage 15. By placing one electrode above the eye surface ectoderm and the other below the embryo (Figure 2A), DNA was introduced into a high percentage of lens epithelial or fiber cells (Figure 3 and Figure 4). Strong EGFP expression was observed as early as 4 h after electroporation and maintained for as long as 5 days in lens cells. Under the electroporation parameters described (five pulses at 20 V and 50 ms duration), the viability of the chick embryos was over 90%. The electroporation success rate is over 30% in lens epithelial cells and over 50% for fiber cells. In these embryos, at least one third of the cell population expresses high levels of EGFP. Electroporation also worked for corneal epithelial and retinal cells. Thus, we have demonstrated that in ovo electroporation can serve as an effective method for introducing DNA into specific types of ocular cells in chicken eyes. We also showed that expressing cDNA encoding a tumor suppressor protein, merlin, in lens epithelial cells, inhibits cell proliferation. This approach offers us a powerful tool to manipulate gene expression during chicken lens development.
We were able to target a group of lens cells at a specific region by slightly adjusting the electroporation method. For example, by placing the electrodes slightly away from the visual axes, we electroporated only half of the epithelial or fiber cells (data not shown). The non-transfected half serves as an internal control in the experiment. Additionally, with a single injection both lens epithelial and fiber cells could be transfected when the electric pulses were applied, and after switching the polarity of the electrodes, reapplied (data not shown). Furthermore, we could perform in ovo electroporation in the lens of chicken embryos older than stage 15 (e.g., stage 20) with a lower efficiency, before the lumen of the lens vesicle is filled with the elongated fiber cells. Therefore, it might be possible to manipulate the expression of a gene, either activating or silencing it, during this narrow time window of lens development. We have not tried to electroporate the lens cells after the lens vesicle lumen is sealed. In theory, DNA can be loaded in the anterior chamber or the vitreous cavity and electroporated into the lens epithelial or fiber cells, respectively. We speculate that the efficiency might be low and expression may not be restricted to only the lens cells.
It is unclear why the electroporation efficiency is lower in lens epithelial cells than in fiber cells. We have considered the following possibilities: (1) Electroporation uses electric field pulses to transiently disrupt the plasma membrane and to drive DNA through the pore of the membrane. It is possible that lens epithelial cells are more resistant, thus affecting the efficiency of electroporation. (2) At stage 15, the anterior lens vesicle cells are actively dividing, while the posterior cells have become post-mitotic and are differentiating into fiber cells. Because the plasmid DNA is not incorporated into the host chromosomal DNA, it may get degraded or diluted during lens epithelial cell proliferation. If one wishes to establish stable misexpression of a transgene in chicken lens, a retrovirus vector containing the gene can be electroporated into cells [42,43]. By this method, introduced DNA will incorporate into the host genome by the integrase activity encoded in the vector. It should be noted that injecting plasmid DNA into the lumen of the chicken lens vesicle can result in EGFP expression in a few fiber cells through diffusion . In our system, we did not observe any EGFP positive cells other than the ones we deliberately targeted. For instance, in all the embryos we targeted for epithelial cell expression, we did not see any isolated EGFP positive cells in the fiber mass, and vice versa. Therefore, we conclude that the efficiency of EGFP expression through diffusion was extremely low in our system.
An alternative effective gene transfer method used in chicken embryos is viral infection [45,46]. A replication competent chicken retrovirus RCAS(A) has been used to express connexin , bcl-2 , and noggin  in developing chicken lenses. Other viral vectors, including replication deficient adenovirus  or retrovirus , were also reported for gene transfer in chicken lens. The percentage of infected lens cells using replication deficient viruses was quite low [49,50]. In comparison to the viral mediated gene transfer methods, electroporation offers several advantages: (1) Gene expression is turned on more quickly after manipulation. In our experiments, EGFP was detected in 4 h, instead of 18-24 h for viral infection. The lens and eye development progresses rapidly at early stages and gene regulation occurs in a very narrow time window. Therefore, to manipulate gene expression it is crucial to turn on the expression of the exogenous gene at desired time points. (2) In electroporation, two or more genes can be simultaneously introduced into cells with no apparent restriction on the size of cDNA insert . (3) No virus production in culture is needed, which minimizes the bio-safety issues and production time. Thus, the electroporation method overall provides a strong alternative approach to analyze gene function in chicken lens and eye development.
In ovo electroporation has been used successfully, mostly for gain of function studies, either over-expressing or misexpressing a gene of interest in a target tissue during chicken embryonic development. Yasuda's group demonstrated that misexpression of L-Maf in the head ectoderm can induce δ-crystallin expression in the surface ectodermal cells outside the lens region [34,52]. In our study, we showed that overexpressing chicken merlin in lens epithelium inhibits cell proliferation. This method could also be very useful to test how different signaling molecules and pathways work together to control cell fate and behavior during embryonic development. In comparison to the transgenic mouse approach, in ovo electroporation requires minimal time, cost, and labor to analyze gene function in vivo.
Thus far, the mouse is still a highly favorable vertebrate model system to conduct loss of function studies, either by expressing a dominant negative form of a protein in transgenic mice or by targeted knock out in ES cells. While making gene "knockouts" has been problematic in chick embryos, new methods have been developed to perform knockdowns of gene expression by in ovo electroporation. Three approaches, including expressing a dominant negative form of the protein , RNA interference (RNAi) [30,32], or morpholinos  have been used with good success.
In summary, we have shown that it is feasible to introduce DNA into chicken lens, corneal, and retinal cells by in ovo electroporation. This protocol has several advantages over the existing viral infection methods used in chicken embryos, such as rapid expression and no size limitation of the DNA insert. In comparison to the transgenic mouse technique, in ovo electroporation requires less time and money. Therefore, this technique provides new opportunities to investigate the molecular regulation of lens and eye development.
This work is supported by NIH grant EY13146 (LWR) and MH59894 (CEK), NIH/NEI core grant EY14795, a grant from Muscular Dystrophy Association (CEK), and departmental unrestricted grant from Research to Prevent Blindness (RPB) Inc. We thank Dr. David Beebe at Washington University School of Medicine for the anti-αA-crystallin antibody, and his valuable comments and advice, Dr. Paul Overbeek at Baylor College of Medicine for his critique on the manuscript, and Dr. Sinead O'Connell and the University of Missouri Molecular Cytology Core for technical support.
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