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Keywords:

  • misexpression;
  • knock-down;
  • neural tube;
  • transposon;
  • RCAS vector;
  • Tet-on and Tet-off

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Principle
  5. Procedure
  6. Results and discussion
  7. References

Gene transfer by electroporation is an indispensable method for the study of developmental biology, especially for the study using chick embryos. Here we briefly review the principles of the method, and its application to chick embryos. Methods of transient misexpression and long-term misexpression by retrovirus vector or transposon system, and knockdown by small interference RNA are reviewed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Principle
  5. Procedure
  6. Results and discussion
  7. References

In the field of experimental embryology, the chick embryo has been a good experimental material because it is easy to get a lot of embryos and easy to access for manipulation during embryogenesis. However, since the main current of developmental biology shifted to molecular developmental biology, the importance of chick embryos as a model animal in developmental biology diminished because of difficulty in gene manipulation in the chick. Establishment of gene transfer in ovo by electroporation revived chick embryos as a model animal in developmental biology (Muramatsu et al. 1997; Sakamoto et al. 1998; Funahashi et al. 1999; Nakamura et al. 2000), and has contributed greatly to our understanding of the molecular mechanisms of development (Nakamura 2001). It is time consuming to establish transgenic or knockout mice, but by electoporation, the area and time of transfection are highly controllable. Furthermore, electroporation of small interference RNA (siRNA) vectors made it possible to knockdown the gene of interest conveniently (Katahira & Nakamura 2003). Here, we review the application of some of these methods in chick embryo.

Principle

  1. Top of page
  2. Abstract
  3. Introduction
  4. Principle
  5. Procedure
  6. Results and discussion
  7. References

According to the explanation of BTX Inc. (1994), cells in liquids can be regarded as a structure consisting of a non-conducting membrane with aqueous solutions on both sides. Exposure to an electric field leads to charge separation in membrane, resulting in a transmembrane potential difference. Opposite electrical charges on the membrane attract each other, exerting a pressure on the membrane, which causes thinning of the membrane. Beyond critical potential difference, small pores are made on the membrane. Through the pores, molecules such as DNA can enter the cells. If the strength of the pulse length and duration are appropriate, removal of the field can lead to healing of the pores. Then DNA enters the nucleus and is transcribed under the enhancer and promoter of the vector.

Procedure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Principle
  5. Procedure
  6. Results and discussion
  7. References

Transient misexpression

cDNA is inserted into the expression vectors that have widely used promoter in mammalian and chicken expression systems. We usually use pRc/CMV (cytomegarovirus) vectors (Invitrogen, Carlsbad, CA, USA), which have a cytomegalovirus enhancer or pMiwIII vector (Suemori et al. 1990; Wakamatsu et al. 1997; Matsunaga et al. 2000) which have a RSV (Rous Sarcoma virus) enhancer and beta-actin promoter.

Long-term misexpression

For long-term expression, the RCAS provirus vector can be used. For this, we have no need to elaborate virus particles, but we have only to electroporate provirus plasmid vector. Widespread misexpression can be obtained by electroporation of the RCAS vector on virus sensitive embryos. On the other hand, electroporation of RCAS vector to virus-resistant embryos allows us to trace the lineage of electroporated cells (Sugiyama & Nakamura 2003). The transposon mediated gene transfer system has recently been adopted, and it assures long-term expression of the transgene (Sato et al. 2007).

Knockdown

For knockdown of certain genes, siRNA is inserted in siRNA expression plasmids (Katahira & Nakamura 2003), which have a U6 promoter or H1 promoter that is suitable for the generation of small RNA transcripts by RNA polymerase III. New vectors that use a chicken U6 promoter driving expression of a modified chicken microRNA were developed (Das et al. 2006). Several companies have improved strategies for selection of siRNA target sequences. In addition, by combining the siRNA with retroviral vectors, stable expression of siRNA can be introduced (Bromberg-White et al. 2004).

Tet-on and Tet-off system

Recently, by using the tetracycline-dependent Tet-on Tet-off system, strict control of expression level and timing has become possible (Hilgers et al. 2005; Watanabe et al. 2007). pBI-EGFP vector and pTRE2 vector for response plasmid that express a gene of interest are used (Hilgers et al. 2005). These plasmids have tetracycline-responsive elements (TRE) combined with minimal CMV promoter, which lack the enhancer that is a part of the complete CMV promoter. This gene expression regulatory element is silent in the absence of binding of tTA or rtTA to tetracycline-responsive element. Tet-on vector expresses reverse tetracycline controlled transactivator (rtTA), binds TRE and activates transcription in the presence of tetracycline (Tc) or the Tc derivative Doxycyclin (Dox). Tet-off vector expresses tetracycline controlled transactivator (tTA), and in contrast binds the TRE and activates transcription in the absence of Tc or Dox. For example, the gene of interest is toxic; tet system is useful because we can control the expression level and timing by changing the administrating volume and timing of Dox.

Electroporation

The plasmid DNA is dissolved in TE (Tris-ethylenediaminetetraacetic acid [EDTA]) buffer. The addition of Fast Green to the DNA solution facilitates visualization. On the second day of incubation, an 18 gauge needle is used to remove a small quantity of albumen from the pointed pole of fertile chicken eggs that have been incubated at 38°C. A window is opened on the top (Fig. 1B), and eggs are staged according to Hamburger & Hamilton (1951). Injection of Indian ink underneath the embryo helps visualize the embryo (Fig. 1C). For the transfection to the neural tube, DNA solution is injected into the lumen of the neural tube with a micropipette (Fig. 1D) made of a glass capillary tube 1 mm in diameter (GD1; Narishige, Tokyo, Japan). Injection of the DNA solution is controlled by mouth. For the transfection to presumptive somatic mesoderm, concentrated DNA solution is placed onto the anterior primitive streak (Sato et al. 2002).

image

Figure 1. In ovo electroporation. After removing 2–4 mL of albumen from the pointed pole of the egg, a pair of electrodes held by a manipulator (A) was inserted from a window opened on the shell (B). Injection of Indian ink underneath the embryo facilitates visualization (C). The electrodes are placed on the vitelline membrane overlying the embryo. It is recommended to drip the Hanks’ balanced solution between the electrodes in order to not injure the embryo by electric pulse. Five electric pulses (25 V, 50 ms) are good for transfection of the neural tube. A pulse of 50 ms is followed by a 950-ms rest phase. DNA solution was injected with a micropipette into the lumen of the neural tube (D). If the DNA solution is injected into the anterior neural tube, making a small hole at the most anterior part of the neural tube helps injection (from Funahashi et al. 1999).

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Various types of electrode pairs supplied by Unique Medical Imada (Sendai, Japan) are used according to the embryonic stage and tissues. For transfection of the anterior neural tube of stage 10 embryos, two parallel stick type electrodes (0.5 mm in diameter, with 1 mm exposure, and fixed 4 mm apart, Fig. 2A) are set, and three to five rectangular pulses (25 V, 50 ms/s, each) are charged (Fig. 1A,C). Transfection to the expanded neural tube (E3–5, mesencephalon; Fig. 2D) is achieved by electroporation (5–10 V, 50 ms, two to three times) using a stick type electrode as a cathode (Fig. 2B), which is placed underneath the left side of the head and a circular electrode is used as an anode (Fig. 2C) and placed on the right side. Effective transfection to somites could be achieved by electroporation at HH stage 7–8 using a stick type cathode (2 mm exposure, Fig. 2B,F,F′) positioned under the embryo and a tungsten microelectrode (Fig. 2E,F,F′) and an anode positioned on the embryos (5–8 V, 25 ms, two to three times) (Fig. 2F,F′, Sato et al. 2002). In general, lower voltage is used when distance between the electrodes is shorter and narrower exposure to electrodes makes it possible to transfect narrower regions (Sugiyama & Nakamura 2003). After electroporation, the window is sealed with Scotch tape, and the embryos are re-incubated at 38°C until the desired stage.

image

Figure 2. Various types of electrodes. Parallel stick type electrodes (1 mm exposure) are routinely used to transfect the neural tube around stage 10. For transfection to the expanded mesencephalon a circular electrode (C), which is placed on the right side of the head is used as an anode and a stick type electrode of longer exposure (B, 2 mm exoposure), which is placed underneath the left side is used as a cathode (D), then charged two to three times (5–10 V, 50 ms). For transfection to the somite, a stick type electrode (2 mm exposure; B) is used as an anode and positioned under the embryo, and a tungsten microelectrode (E) is used as a cathode (5–8 V, 25 ms, two to three times) and put on the embryo at the HH stage 7–8 (F). Position of electrodes and plasmid for transfection to somite is schematically shown (F and F′); dorsal view (F) and transverse section (F′). Phosphate buffered saline (PBS) or Hanks solution (indicated by blue on F and F′) is put on the caudal part of the embryo, and DNA solution is put on the primitive streak (green on F and F′), where gastrulation is taking place (circles on F′ indicate gastrulating cells). di, diencephalons; ep, epiblast; hy, hypoblast; me, mesoderm; met, metencephalon.

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Doxycycline injection: After electroporation of Tet vectors, 20 µg/mL doxycycline/PBS is injected underneath the embryo by micropipette (3–5 µL). Stronger expression can be obtained with increasing volumes of doxycycline.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Principle
  5. Procedure
  6. Results and discussion
  7. References

By electroporation, negatively charged DNA could be introduced into the cells at the anode side. Co-electroporation of the reporter gene, such as green fluorescent protein (GFP) makes it possible to assess the efficiency of transfection in ovo (Fig. 3C). By 2 h after electroporation, translation product is detectable (Fig. 3A, Funahashi et al. 1999), and the expression level increases gradually (Fig. 3B,D). Beyond 24 h after electroporation, the expression level decreases gradually, but lacZ expression could still be observed strongly after 72 h of electroporation (Fig. 3F). LacZ product could still be detected 5 days after electroporation (Katahira et al. 2000).

image

Figure 3. Efficiency of electroporation. Efficiency of transfection could be assessed in ovo by coelectoporated green fluorescent protein (GFP) expression (C, 9 h after electroporation). The translation products of lacZ can be recognized 2 h after electroporation (A), and more products are seen as time elapses until 24 h after electroporation (B: 3 h after electroporation; D: 24 h after electroporation). As DNA is negatively charged, DNA moves toward the anode side so that the DNA is introduced to the anode side. At 24 h after electroporation (D, E), more than half of the cells express lacZ in the transfection field. The lacZ translation products are still detectable at 72 h after electroporation (F). (from Funahashi et al. 1999).

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Gene silencing by electroporating short hairpin RNA (shRNA) has been realized (Katahira & Nakamura 2003; Nakamura et al. 2004). It has been shown by siRNA against En2 that interference could be obtained up to a two nucleotide mismatch, but that four nucleotide mismatches completely abolished interference (Fig. 4; Katahira & Nakamura 2003).

image

Figure 4. Effects of small interference RNA (siRNA). Whole-mount in situ hybridization for En2 (A, B, G, H) and En1 (D, E). Photographs on the same row are from the same embryo. Figures of the left column show the control side of the embryo (A, D, G), and figures of the right column show green fluorescent protein (GFP) fluorescence in the transfected side (C, F, I). For A–F, shRNA is prepared to interfere with En2 expression, and has 2 base pair mismatch to En1. The electroporated short hairpin RNA (shRNA) interfered with En2 expression strongly (A, B), and with En1 weakly (D, E). Sequences that are prepared as 4-base mismatches to En2 (G–I) did not interfere with En2 expression (Compare G and H) (from Katahira & Nakamura 2003). mes, mesencephalon; met, metencephalon.

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Using the Tet-on, Tet-off system, we can regulate gene expression temporally. An example of the use of Tet-on vector and response vector that can regulate GFP is shown in Figure 5. These vectors are transfected to the spinal cord by electroporation in chick embryos. Translation product is not detectable at Dox administration (21 h after electroporation) (Fig. 5A), 5 h after Dox administration, that is 26 h after electroporation, sufficient expression of GFP could be seen (Fig. 5A′), and stronger expression could be obtained at 30 h after dox administration (Fig. 5A′). Using the Tet-on and Tet-off system, we can regulate expression of a gene of interest, but it must be remembered that long-term expression could not be obtained by the usual plasmid expression vectors. With the recently developed transposon system we can obtain long-term expression of transferred genes (Sato et al. 2007), and the combination of Tet-on and Tet-off with the transposon system has enabled long-term regulation of the expression of the transferred gene (Watanabe et al. 2007).

image

Figure 5. Regulation of expression by Tet-on system and long-term expression by RCAS. Tetracycline controlled expression of electroporated green fluorescent protein (GFP) in neural tubes of chick (A, A′, A″). We co-electroporated pBI-enhanced green fluorescent protein (EGFP) vector and pCAGGS-rtTA vector at stage 13. At 21 h after electroporation, when Dox was administrated, GFP fluorescence could not be observed (A). At 5 h after Dox administration, GFP was expressed sufficiently (A′), and GFP signal increased at 30 h after Dox administration (A″). (B) GFP expression in oculomotor nerves (arrows). Electoporation was carried out at E2, Dox was administrated at E3, and fixed at E4. (C) Long-term expression by electroporation of RCAS-virus vector on virus sensitive embryos. Electroporation to mesencephalon was carried out at E2.5. At E8.5, the chick mesencephalon consists of six layers (D), and cells in all layers of mesencephalon are labeld by GFP (C). VL, ventricular layer.

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For long-term expression, RCAS vector is effective. If we use virus-sensitive embryos, RCAS virus produced by transfected cells infects adjacent cells, and we can misexpress a gene of interest in cells in a wide area (Fig. 5C). In contrast, if we electroporate on virus-resistant embryos, virus produced by transfected cells cannot infect adjacent cells, and transfection is limited to the descendents of the transfected cells. Thus we can trace the lineage of the transfected cells (Sugiyama & Nakamura 2003).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Principle
  5. Procedure
  6. Results and discussion
  7. References