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Keywords:

  • chick;
  • in ovo electroporation;
  • reitna;
  • retrovirus vector

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Procedure for in ovo electroporation
  5. Electroporation of the replication-competent retroviral vector
  6. Conclusion
  7. References

Owing to its external position in the embryo, the chick eye has been used as a readily accessible model for studying the molecular mechanisms behind the patterning of the central nervous system. Although methods of genetic analysis have not been established as in the mouse, the chick is convenient for analyzing the functions of genes by in ovo electroporation of retroviral vectors. In this review, we describe the retroviral vector-mediated transfer of genes into the chick optic vesicle by in ovo electroporation. A rapid, efficient, and sustained expression of transgenes is achieved by this approach.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Procedure for in ovo electroporation
  5. Electroporation of the replication-competent retroviral vector
  6. Conclusion
  7. References

The chick embryo offers many advantages for developmental studies over other vertebrate embryos as it allows easy access for in ovo surgical manipulations, such as tissue transplantation and the implantation of cultured cells or chemically treated beads for the local release of humoral factors. In particular, owing to its external position in the embryo, the chick eye is a popular model for studying the patterning mechanism of the central nervous system (CNS). This patterning has a crucial role in shaping functional organization because it is the basis of the specific wiring in the CNS. Genetic analysis is not easy in the chick, as compared with the mouse for which transgene introduction or gene targeting techniques have been well established. However, because methods for the expression of exogenous genes and for gene silencing in the chick embryo have been recently developed, the functional analysis of genes has become possible in combination with classical techniques of developmental biology and neurobiology.

One general method is replication-competent retrovirus particle-mediated gene transfer in the chick embryo (Morgan & Fekete 1996). After the infection of viral particles, the viral RNA is reverse-transcribed and integrated into the host genome. Moreover, because the replication-competent viral vectors harbor all of the genome sequences essential for the production of infectious viral particles, horizontal infection continues to the neighboring cells in the host through reproduction of viral particles. Thus, the stable and efficient expression of a transgene can be achieved by this method. However, the system has several restrictions. For example, only coding sequences of less than 2.5 kb can be packaged into the vector. Also, it takes over 24 h for the proteins encoded in the retroviral vectors to be adequately expressed after the injection of viral particles. Therefore, this method is not suitable for the functional analysis of genes in the early developmental stages.

The in ovo electroporation of expression plasmids is another way to transfer genes into chick embryos (Muramatsu et al. 1997; Funahashi et al. 1999; Yamagata et al. 1999). This system has the advantage that multiple genes can be simultaneously introduced into embryos with no apparent restriction in the size of the transgenes. However, the expression of transgenes is transient, lasting for only a few days even though the expression is driven by efficient promoters such as pMiwClaI (Yamagata et al. 1999) and pCAGGS (Niwa et al. 1991). This failure to achieve a stable expression is attributable to the inability of plasmids to be incorporated in the embryo: they are not integrated into the host genome, and as a result, become diluted as embryonic cells proliferate.

As a third method to overcome these drawbacks, the in ovo electroporation of retroviral vector DNA was developed. With this method, the rapid, efficient, and sustained expression of a transgene is achieved, because the mRNA is directly transcribed from the introduced retroviral vector DNA shortly after the gene transfer, which is followed by the production of viral particles and continuous widespread infection. Here we describe the retroviral vector-mediated transfer of genes into the chick optic vesicle by in ovo electroporation.

Procedure for in ovo electroporation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Procedure for in ovo electroporation
  5. Electroporation of the replication-competent retroviral vector
  6. Conclusion
  7. References

Chick strain

The avian sarcoma-leukosis virus (ASLV) family is classified into five major subgroups, A to E, by the envelope protein (Hughes 2004): RCAS vectors are derived from Rous sarcoma virus. ASLV-infected chick cells are highly resistant to reinfection by ASLVs of the same subgroup. Chick strains differ in their susceptibility or resistance to different subgroups due to the endogenous proviruses that are closely related to the ASLV. Therefore, it is necessary to use strains susceptible to the subgroup of the retroviral vector. A chick strain susceptible to all five subgroups (C/O) is now available commercially.

Plasmids

We usually use a replication-competent retroviral vector, RCAS-NS (Fig. 2A; Suzuki et al. 2000), which is a modified vector of subgroup B RCASBP (Hughes et al. 1987). The transgene was transferred to RCAS-NS after the coding region was cloned once into the shuttle vector SLAX-NS (Suzuki et al. 2000). SLAX-NS has the 5′-untranslated region of the src gene, which confers efficient expression on heterologous coding sequences (Morgan & Fekete 1996). Plasmids are prepared using the Qiagen Plasmid Kit (Qiagen, Hilden, Germany).

image

Figure 2. Rapid and sustained expression of enhanced green fluorescence protein (EGFP) after electroporation. (A) Schematic representation of the RCAS-NS vector. SA, splicing acceptor; SD, splicing donor; LTR, long-terminal repeat. (B) A stage 9 embryo electroporated with RCAS/EGFP at stage 8. EGFP fluorescence was already detected in the entire right optic vesicle (arrow heads) at 6 h after electroporation. Anterior is upwards. (C, D) Flat mount of E8 and E16 retinae transfected with RCAS/EGFP. EGFP fluorescence was continuously detected in the entire retina at E8 (C) and E16 (D). Temporal is left, and dorsal is upwards. (E) Coronal section of E8 retina transfected with RCASDC-CU6/BMP2 at E1.5. The whole of the right retina was infected. Viral gag protein was visualized at E8 by immunostaining with the monoclonal antibody 3C2. The boxed regions in the control retina (left) and manipulated retina (right) are enlarged on the right-upper and right-lower side, respectively. D and V indicate dorsal and ventral, respectively. Bars: (B) 100 µm; (C) 2 mm; (D) 3 mm; (E) 1 mm. Modified from Sakuta et al. (2006) (E).

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Preparation of the embryos

Fertilized eggs are incubated horizontally at 37.5°C in a humidified incubator until they reach Hamburger Hamilton stage 8–10 (Hamburger & Hamilton 1951), 26–40 h. After sterilization with 70% ethanol, a small hole is made at the sharp side of the egg with fine forceps, and then 3–5 mL of albumen is removed with an 18-gauge needle attached to a 5-mL syringe. After the hole is sealed with plastic tape, the top of the horizontal shell is cut elliptically with small scissors to open a window over the embryo. If the embryos have not reached stage 8–10, the eggs are further incubated at 37.5°C after the window is sealed with plastic tape.

Electroporation

A pair of platinum electrodes (CUY611P3-1; Unique Medical Imada, Miyagi, Japan) is set with a micromanipulator 4 mm apart. After the window of the shell is opened, the embryo is made wet with about 200 µL of Hanks’ saline. The cathode and anode are placed on the vitelline membrane on the left and right sides of the rostral region of the embryo, respectively (Fig. 1A,B), and wetted with about 200 µL of Hanks’ saline, so that the resistance between electrodes decreases to around 1.0 kΩ. A microglass pipette filled with a DNA solution is inserted into the anterior neural fold at stage 8 (Fig. 1A) or the optic vesicle at stage 9–10 (Fig. 1B), and the DNA solution is injected by mouth. The microglass pipettes for injection of the DNA solution into the embryo are made from glass capillary tubes 1 mm in diameter (GD-1; Narishige, Tokyo, Japan) using a puller (tip diameter; about 40 µm). About 0.2–0.4 µL of DNA solution in phosphate-buffered saline (PBS) containing 0.05% fast green is injected into individual embryos. The concentration of DNA is 0.1–2.0 µg/µL. After injection of the DNA solution, electric square pulses (20–24 V, 50 ms) are applied by a CUY21EDIT electroporator (BEX, Tokyo, Japan) three to five times at an interval of 400 ms. After electroporation, the window is sealed with plastic tape, and the embryos are reincubated at 37.5°C until they reach the desired developmental stage.

image

Figure 1. In ovo electroporation into the chick optic vesicle. (A, B) Schematic representation of in ovo electroporation. A pair of platinum electrodes (0.5 mm in diameter with an exposed length of 1 mm) is set with a micromanipulator 4 mm apart. The cathode and anode are placed on the vitelline membrane on the left and right sides of the rostral part of the embryos, respectively. To transfect the eye region, the DNA solution is injected into the anterior neural fold at stage 8 (A) or the optic vesicle at stage 9–10 (B). DNA moves from the cathode toward the anode in the electric field (red arrow) and the right (anode) side of the embryo is selectively transfected. (C) Evaluation of transfection efficiency. An E3 embryo electroporated with RCAS/AP carrying the human placental alkaline phosphatase gene and pEGFP-N1 at E1.5 (left). The same embryo was observed for enhanced green fluorescence protein (EGFP) fluorescence, visualizing the right retina expressing the transgenes (right). Bar, 1 mm.

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Because DNA is negatively charged, it moves from the cathode toward the anode in the electric field, and only the right (anode) side of the embryo is transfected. The left (cathode) side of the embryo serves as a control. Therefore, you can compare the effects of the manipulation in the same embryo.

Electroporation of the replication-competent retroviral vector

  1. Top of page
  2. Abstract
  3. Introduction
  4. Procedure for in ovo electroporation
  5. Electroporation of the replication-competent retroviral vector
  6. Conclusion
  7. References

Evaluation of transfection efficiency

After electroporation, the chick embryo is repositioned to the original right-side up orientation. To monitor the transfection efficiency in ovo under a fluorescence stereoscopic microscope, coelectroporation of an expression plasmid for a marker protein is useful. For this purpose, we usually use an expression plasmid for the enhanced green fluorescence protein (EGFP), pEGFP-N1 (Clonetech, Palo Alto, CA, USA). We usually check the transfection efficiency at E3 in ovo (Fig. 1C) and discard the embryos of low transfection.

Expression of a transgene after electroporation

We have electroporated RCAS/EGFP as a model into the anterior neural fold at stage 8. EGFP fluorescence is already detectable in the entire right optic vesicle at 6 h after electroporation (Fig. 2B), as early as stage 9, just before the stage when the regional specificity along the anteroposterior axis is reportedly determined (Dütting & Meyer 1995; Dütting & Thanos 1995). When a conventional expression vector, such as pMiwClaI and pCAGGS, is electroporated, the expression of the transgene is transient, lasting merely 2–3 days. In contrast, electroporation of the retroviral vector leads to integration of the provirus into the host genome and a long-lasting expression. As expected, EGFP fluorescence was detected in the entire retina at E8 (Fig. 2C), and still at E16 (Fig. 2D) when the retinotectal map is already established (Nakamura & O’Leary 1989). A higher level of expression of a transgene just after the electroporation can be achieved by coelectroporation of an RCAS-NS vector together with a conventional expression vector carrying the same transgene. The rapid and sustained expression achieved by this method is effective for analyzing the functions of a gene at the early developmental stages (Sakuta et al. 2001; Takahashi et al. 2003).

Because the DNA concentration of a retroviral vector necessary for efficient infection varies depending on the transgene, the optimal concentration must be examined experimentally. The efficiency of retroviral infection can be evaluated by visualizing the expression of the transgene and viral genes. We usually check the efficiency of infection by immunostaining the viral gag protein using the monoclonal antibody 3C2 (Potts et al. 1987). When the RCAS vector was electroporated under optimal conditions, the whole of the right retina was infected at E8 (Fig. 2E).

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Procedure for in ovo electroporation
  5. Electroporation of the replication-competent retroviral vector
  6. Conclusion
  7. References

We can now achieve the rapid, effective, and constitutive expression of a transgene in the chick embryo using retroviral vector-mediated transfer by electroporation. In addition, the size limit of the replication-competent retroviral vector can be circumvented by using a replication-incompetent vector (Shintani et al. 2006), and the temporal expression of a transgene can be manipulated by the tet regulatory system (Sakuta et al. 2006). Furthermore, electroporation of a retroviral vector carrying a short hairpin RNA-expression cassette is an effective way to induce sustained gene silencing in the chick embryo (Sakuta et al. 2006; Shintani et al. 2006). We hope that these methods help to revive use of the chick as a model animal in developmental biology and neurobiology.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Procedure for in ovo electroporation
  5. Electroporation of the replication-competent retroviral vector
  6. Conclusion
  7. References