Abstract
Preimplantation embryos of mammals are enclosed by a translucent layer called zona pellucida (ZP), which is composed of glycoproteins. ZP is important for protecting against infection by virus and bacteria, and to prevent attachment of embryos to the oviductal epithelia. Due to the presence of ZP, it has been difficult to transfect preimplantation embryos existing within the oviductal lumen, with exogenous nucleic acids, such as DNA and mRNA. However, intraoviductal instillation of nucleic acids, and subsequent in vivo electroporation in pregnant females, enables transfection of these embryos, leading to the production of gene-modified animals. This new method for production of genetically modified animals does not require any ex vivo handling of embryos, which has been essential for traditional transgenesis. In this article, we describe recent advances in the in vivo transfection of preimplantation mammalian embryos, and also the possibility of simple transfection of these embryos through intraoviductal instillation of a solution, alone.
Keywords
- genome editing
- GONAD
- in vivo gene transfer
- oviduct
- preimplantation embryos
1. Introduction
Preimplantation embryos present within the oviductal lumen would be the most favorable targets for genetic manipulation, since the oviducts themselves can be easily exposed outside the individual, under anesthesia, and manipulation within this organ would be possible under observation, using a dissecting microscope. Genetic material can be introduced into zygotes or cleaving embryos, floating in the oviductal lumen, by inserting a glass pipette through the oviductal wall, and introducing genetic material successfully into these embryos by penetrating the zona pellucida (ZP). If this event occurs,
The ZP is a multilayered porous matrix of glycoproteins that envelopes mammalian oocytes and preimplantation embryos, protecting them from environmental insults, including viral infection, and injury by chemical or physical substances [8]. Therefore, it has been difficult to transfect mammalian oocytes and preimplantation embryos, with the usual transfection methods that have proven useful for somatic cells. Early attempts at gene delivery to preimplantation embryos involved the transfection of ZP-free embryos. However, such embryos are vulnerable, adhesive, and easily damaged [9]. The most commonly method used for penetration of ZP for gene delivery is the pronuclear microinjection using purified DNA [10], or the microinjection of viral elements into the perivitelline space, between ZP and the zygotes [11]. Furthermore, it is possible to transfect mouse fertilized eggs with lentiviral vectors,
In the present article, we first discuss studies that have reported successful delivery of substances to ZP-enclosed embryos
2. Transfer of exogenous substances into ZP intact embryos via simple incubation
As mentioned previously, it may be possible to perform gene delivery to embryos if a substance capable of penetrating ZP is used in combination with nucleic acids. For example, Ivanova et al. [16] demonstrated that when ZP intact preimplantation embryos from mice and rabbits were incubated in a medium containing DNA-carrying constructs with insulin as an internalizable ligand, (insulin-polylysine)-DNA and (insulin polylysine)-DNA-(streptavidin-polylysine)-(biotinylated adenovirus), the constructs penetrated the ZP and accumulated within each blastomere. Southern blot hybridization revealed chromosomal integration of transgenes in mid-gestational fetuses and in a newborn. The ligand-mediated gene delivery to early embryos can be explained by the following mechanism. Insulin provides delivery inside the cell, while adenoviruses ensure release from the endosomes. They called this type of gene delivery “receptor-mediated gene transfer”. According to the authors, the construct containing DNA and insulin, penetrates inside, to accumulate in the peri-nuclear space of the embryos. Munk et al. [17] demonstrated that multiwall carbon nanotubes (MWNTs) could cross the ZP to help the delivery of plasmid DNA into bovine embryos
As mentioned previously, EP enables the delivery of exogenous substances to somatic cells and ZP-enclosed embryos. Grabarek et al. [13] was the first to demonstrate that nucleic acids can be efficiently delivered to isolated embryos (oocytes and zygotes) by EP. Recently, Kaneko et al. [26] first demonstrated successful induction of genome editing of the target locus in rat embryos, when they were electroporated
3. Intraoviductal instillation of a solution
Delivery of liposomally encapsulated DNA directly into the oviductal lumen was first reported by Esponda’s group [30, 31]. The purpose of their study was to transfect epithelial cells lining the oviductal lumen, not to deliver genetic materials to preimplantation embryos floating in the oviductal lumen. They found that ~6% of oviductal epithelial cells were successfully transfected. Sato [32] injected a solution containing plasmid DNA [conferring enhanced green fluorescent protein (EGFP) expression] into the oviductal lumen of pregnant female mice on Day 0.4 (~11:00 h; corresponding to early zygotes; the day when the vaginal plugs are detected is designated as Day 0 of pregnancy), and then performed
Figure 1 represents a schematic illustration of the structure of murine oviduct and ovary, on different days of pregnancy; Days 0.4 (Figure 1a), 0.7 (~16:00 h; corresponding to late zygotes; Figure 1b) and 1.4 (~1d, 11:00 h; corresponding to 2-cell embryos; Figure 1c), are illustrated. After mating with males, ovulated oocytes, tightly surrounded by cumulus cells, are transferred to the specific site of the oviduct - the ampulla- following which fertilization occurs at this site. On Day 0.4, zygotes (fertilized eggs) are still enclosed by cumulus cells and exist at the ampulla. On Day 0.7, zygotes are still present at the ampulla, but the ampulla itself, exhibits shrinkage and detachment of cumulus cells from the zygote. On Day 1.4, zygotes cleave to form 2-cell embryos, and are present at the ampulla or the oviductal segment between the ampulla and the isthmus.
In Figure a schematic illustration of the moments before (Figure 1d) and during (Figure 1e) intraoviductal instillation of a solution is provided. Sato et al. [33] injected a solution containing Hoechst33342 dye, frequently used for vital nuclear staining, into the oviductal lumen of pregnant females at Day 0.4. One day after the surgery, 2-cell embryos were collected from the oviducts for checking the incorporation of the dye in their nuclei. They observed that all the collected embryos had fluorescent nuclei. This means that the dye injected into the oviductal lumen penetrates the ZP, enters the zygotes, and binds to their nuclear DNA. Here, we examined whether another dye 4′, 6-diamidino-2-phenylindole (DAPI), also used for staining of nuclei, can also bind to nuclear DNA of zygotes
4. Gene delivery into preimplantation embryos in vivo
To our knowledge, successful gene delivery into preimplantation embryos
Takahashi et al. [34] employed the method of Sato et al. [33] to induce genome editing in the target gene (
As mentioned above, GONAD on Day 1.5 results in production of individuals with highly frequent mosaic mutations. Genome editing at 2-cell embryo stage, appears to be the major cause of this problem. To circumvent the issue of mosaic mutations, performing GONAD on Day 0.7, a stage corresponding to late 1-cell embryos, from which cumulus cells begin to detach (see Figure 1b), was considered. Ohtsuka et al. [35] examined whether embryos at this stage are suitable for GONAD by injecting a solution containing
5. Genome editing in preimplantation embryos after performing GONAD on day 0.7
Next, we tested whether GONAD can indeed induce specific mutations on the endogenous target locus in late 1-cell embryos. We chose α-1, 3-galactosyltransferase (α-GalT) gene (
We designed gRNA (5′-GAGAAAATAATGAATGTCAA-3′) for targeting exon 4 of murine
6. Improved (i )-GONAD in mice
As already mentioned in Section 4, Ohtsuka et al. [35] modified GONAD as follows: 1) RNP was employed instead of Cas9 mRNA and gRNA; and 2)
Administration of higher dose (in this case, more than 5 IU) of gonadotrophins can induce superovulation, but often causes failure to deliver pups [43, 44, 45]. Some reports state that the administration of low-dose gonadotrophins (less than 5 IU) facilitates the ovulation of a natural number of oocytes, and successful delivery of pups [46, 47]. This evoked us to suppose that the use of females who have achieved pregnancy after administration of low-dose gonadotrophins and subsequent mating with males would be preferable for
Next, we tested whether genome-edited live mid-gestational fetuses can be obtained by
7. Conclusion
Since preimplantation embryos, including embryos at stages of fertilized eggs to cleaved eggs, are floating in the oviductal lumen, it is easy to manipulate them through introduction of substances (such as genome editing-related components, plasmid DNA, viral vectors and chemical reagents) into the oviductal lumen. Particularly, intraoviductal instillation of genome editing-related components and subsequent
Acknowledgments
This study was partly supported by a grant (no. 24580411 for M.S.; no. 16H05049 and 16 K15063 for S.N.) from the Ministry of Education, Science, Sports, and Culture, Japan.
Conflicts of interest
The founding sponsors had no role in the design of the study, collection, analyses, or interpretation of data, writing of the manuscript, and decision to publish the results.
Author contributions
Masahiro Sato designed the study and drafted the manuscript; Masato Ohtsuka provided information of
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