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Keywords:

  • chick embryo;
  • gene transfer;
  • in ovo electroporation;
  • in utero electroporation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. In ovo electroporation
  6. Extension to other systems
  7. Further extension
  8. References

Gene transfer by electroporation has become an indispensable method for the study of developmental biology. The technique is applied not only in chick embryos but also in mice and other organisms. Here, a short history and perspectives of electroporation for gene transfer in vertebrates are described.


Background

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. In ovo electroporation
  6. Extension to other systems
  7. Further extension
  8. References

The chick embryo has been a cornerstone of experimental embryology since it is easily accessible for manipulation during embryogenesis, and this model system has contributed greatly to our understanding of tissue interactions during development. In particular, transplantation experiments between chick and quail have enabled close monitoring of the fate of long migrating cells during development (Le Douarin 1982, 2008). However, production of transgenic chicken and gene targeting experiments have been difficult in the chick, because the embryos remain in the oviduct and uterus for the first 24 h of embryogenesis, which has limited analyses of gene function, and led to a gradual decrease in interest in this developmental model.

Among the gene transfer systems, electroporation has become one of the most popular systems both in vivo and in vitro because of its simplicity and efficiency. The principle of electroporation is that small pores are made on the membrane by an electric field. Through the pores, charged 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 (Funahashi et al. 1999). At first, electroporation was carried out at high voltage so that it was limited to bacteria transformation, since tissues of higher organisms are damaged by high voltage. Nevertheless, electroporation in higher organisms was carried out for the transfer of drugs for the treatment of cancer; bleomycin was successfully applied to hepatocellular carcinoma in Donryu Rat (Okino & Mohri 1987).

In ovo electroporation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. In ovo electroporation
  6. Extension to other systems
  7. Further extension
  8. References

Electroporation at first was carried out by high voltage and short pulse. Since high voltage damages the tissue, mild conditions had been pursued by late Dr Muramatsu, Nagoya University, and Mr Hayakawa, Nepa Gene, since 1994. They tried electroporation by low voltage, long square pulse, and found 5–8 times of 20–100 V, 20–50 ms pulses successfully transfected adult chicken oviduct (Ochiai et al. 1998). They had also tried and successfully transferred genes to chick embryos in ovo, and they presented a poster in the joint annual meeting of the Japanese Biochemical Society and the Molecular Biology Society of Japan in 1996, and published a paper in 1997 (Muramatsu et al. 1997).

We were shocked by their poster showing highly efficient transfection in somites, and decided to establish stable high efficiency of misexpression of certain genes in ovo. Mr Imada (Unique Medical Imada, Natori, Japan) helped us to find the best conditions to transfect chick embryos in ovo. At first trials, all the embryos electroprated in ovo died, but an embryo that had received neural tube electroporated in vitro survived and showed very high efficiency of transfection. To see such high transfection (lacZ expression), we were convinced of the success of this method for application in developmental biology. We came to conclude that the best conditions for electroporation to the mesencephalon of stage 10 embryos is five rectangular pulses of 25 V with electrodes of 0.5 mm of diameter and a 1.0 mm exposure being put on the vitteline membrane 4 mm apart (Fig. 1) (Funahashi 1997; Funahashi et al. 1999; Nakamura et al., 2000) (now milder conditions are adopted, Odani et al. 2008). In order to carry out electroporation to chick embryos, we must prepare an electroporator, electrodes, simple micromanipulator and micropipette puller to prepare for micropipettes. It is important that the electroporator can stably emit low voltage rectangular pulses. Electrodes should be prepared to fit the target tissue. The method is so effective that we could study molecular mechanisms of brain regionalization and analyze mechanisms of isthmus organizing signal (Nakamura 2001; Nakamura et al. 2008).

image

Figure 1. In ovo electroporation. After removing 2–4 mL of albumin from the pointed pole of the egg, a pair of electrodes held by a micromanipulator (A) are 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 Hanks' balanced solution between the electrodes in order to prevent drying of the embryo. Five electric square pulses (25 V, 50 ms) is 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 to 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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Extension to other systems

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. In ovo electroporation
  6. Extension to other systems
  7. Further extension
  8. References

Transfection to many other tissues in chick embryos by in ovo electroporation has been achieved (Nakamura 2009). Chick embryos at primitive streak stages are vulnerable, and do not survive electroporation in ovo. Electroporation on chick embryos could be achieved in vitro at low voltage, and the embryo be cultured in a new culture system. (Kobayashi et al. 2002; Hatakeyama & Shimamura 2008; Voiculescu et al., 2008). Electroporation has also been applied to mouse embryos. It was first adopted to transfect mouse embryos in whole mount culture (Osumi & Inoue 2001), and then to trace cell lineages in the mouse brain (Saito & Nakatsuji 2001; Tabata & Nakajima 2001). By in utero electroporation, transfection of mouse embryos was achieved in an area- and time-specific manner, and was applied to the study of telencephalon development (Fukuchi-Shimogori & Grove 2001).

Electroporation has been applied to fish (Chen et al. 2009) and ascidian embryos (Corbo et al. 1997), and to the regenerating tissue (Thummel et al. 2006; Mochii et al. 2007).

Further extension

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. In ovo electroporation
  6. Extension to other systems
  7. Further extension
  8. References

At first, electroporation was very effective for gain of function of transcription factors and signaling molecules. For loss of function of a gene of interest, electroporation of fluorescein conjugated morpholino antisense oligonucleotide was adopted (Kos et al. 2003; Sugiyama & Nakamura 2003). It must be noted that morpholino antisense oligonucleotide interferes with translation, and consequently we need an antibody against the molecule to evaluate the results precisely.

When it was shown that the short hairpin RNA can interfere with the target RNA (Svoboda et al. 2001), we tried to knockdown the target gene by electroporating shRNA expressing vector (Katahira & Nakamura 2003; Odani et al. 2008). This technique allowed us to knockdown the gene of interest at the desired place and time in chick embryos. Now gain- and loss-of function experiments can be achieved conveniently in chick embryos, and chick embryos were revived as model animals for the study of developmental biology.

Recently transposon-mediated gene transfer in chick was developed by Takahashi's group (Sato et al. 2007). They adopted Tol2 transposon, which was isolated from Japanese medaka fish. piggyBack was also shown to be an effective transposon to make transgenic chicken (Liu et al. 2012). In mammalian cell lines, piggyback, TolII and SB11 were shown to be active (Wu et al. 2006). Genes in the transposon vector are integrated into the genome in the presence of transposase so that we can get long-term expression of the transgene. In addition, combination of the Tet-on, and Tet-off system and transposon system made it possible to control the timing of expression of a transferred gene for a longer period (Hilgers et al. 2005; Sato et al. 2007; Watanabe et al. 2007; Dubrulle & Pourquié 2009; Sato & Takahashi 2009). Now this system has become a routine method in the study of developmental biology.

shRNA expression vector is usually driven by RNA polymerase III promoter (Pol III), which is mainly involved in highly expressed infrastructural RNAs, and it is difficult to control its regulation (Dieci et al. 2007). Expression vector for siRNA expression from long double strand RNA (dsRNA) was developed (Shinagawa & Ishii 2003). The vector encodes long double strand RNA (dsRNA) which is expressed by Pol II, and is designed so that cap structure and polyA are deleted from transcribed long dsRNA. Since transcribed dsRNA lacks the cap and poly A tail, it stays in the nucleus, where it is processed into siRNA by ribonuclease(s) such as Dicer. Combining this vector with the Tet-on and -off system, we can control siRNA expression by Tet (Hou et al. 2011). Now siRNA expression vectors, which use pol II, are commercially available.

We can use provirus RCAS retrovirus for electroporation. We need not elaborate on virus particles, but we have only to electroporate provirus RCAS plasmid vector (Sakuta et al. 2008). If we electroporate virus-sensitive embryos, the virus produced in the transfected cells infects adjacent cells and widespread misexpression could be obtained. On the other hand, if we electroporate virus-resistant embryos, adjacent cells are not infected by the virus, and the expression is limited to the descendents of the transfected cells (Sugiyama & Nakamura 2003), and thus we can trace cell lineage.

It is difficult to transfect mesenchymal cells by electroporation, because DNA can easily diffuse after microinjection to loosely associated tissue, and because it is difficult to find luminal space to deposit DNA (Krull 2004). It is proposed that electric resistance of mesenchymal tissue is different from that of epithelial tissue, that the optimal condition for electroporation is different (Krull 2004). Transfection to somites could be achieved by targeting the somitocoele at the epithelial somite stage (Scaal et al. 2004), and by targeting the somite precursor, that is, the gastrulating epiblast (Ohata & Takahashi 2009). Transfection to chick limb buds could be achieved by targeting lateral plate mesoderm of the limb field (Krull 2001; Suzuki & Ogura 2008, 2009).

Perspectives

Uchikawa et al. have been successful analyzing enhancers in chicken embryos by electroporation (Uchikawa et al. 2004; Uchikawa 2008). They electroporated early embryos in New culture. This method is much more convenient than analysis in transgenic mice, and allows enhancer analysis of mouse genes in chick embryos (Lee et al. 2004). But in New culture, it is difficult to analyze organogenesis. The method in which New cultured embryos are returned to the egg, and can be maintained around E5.5 has been developed (Tanaka et al. 2010). Combination of these methods will facilitate the analysis.

Linking of electroporation with time-lapse imaging of embryos in shell-less culture, or of tissues in improved culture systems will contribute to deepen our understanding of cell behavior in the embryo. Culture chambers for shell-less culture and for better imaging has been in preparation in our lab. Saggital explant culture, where embryos are laid on the culture chamber after being shallow cut along the midline of E2 chick embryos, is a good system for observation of neural crest cell migration (Kasemeier-Kulesa et al. 2005).

In utero electroporation to mouse embryos has so far mainly applied to the analysis of the central nervous system. The method has been improved and by the use of special promoter, it is possible to target special type of cells (Nishiyama et al. 2012). The method of electroporation is much more convenient than transgenic or KO mouse production and it will contribute to the study of brain disorders. Such a study has begun (Taniguchi et al. 2012).

Apart from developmental biology, Professor Miyazaki has been successfully carrying out gene transfer into adult muscles by electroporation in order to deliver cytokines, growth factors and other serum factors for basic research and human gene therapy (Aihara & Miyazaki 1998; Miyazaki & Miyazaki 2009). Recently, cytokines have been applied for the treatment of melanoma (Heller & Heller 2010) and vascular endothelial growth factor (VEGF) to induce vasculogenesis for wound healing (Ferraro et al. 2009; Makarevich et al. 2012). Clinical application of electroporation is also a hope for the future (Heller & Heller 2011) .

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Background
  5. In ovo electroporation
  6. Extension to other systems
  7. Further extension
  8. References