東海大学医学部基礎医学系分子生命科学 School of Medicine, Tokai University
Pronuclear Injection-based Targeted Transgenesis (PITT)
In 1980, transgenic (Tg) mice were first created by Gordon et al. who microinjected purified DNA (transgenes) into pronuclei of zygotes [which is thus called “pronuclear injection (PI)”]. Since then, this method has been recognized as a golden standard for creation of Tg animals. Unfortunately, this method has some demerits, as exemplified by random chromosomal integration of multiple copies of transgenes. As a result, variegated expression of transgenes occurs. In some cases, gene silencing of transgene often occurs. Therefore, researchers must produce multiple Tg founders per a transgene, which is labor-intensive and it costly. The most ideal way to produce desirable Tg animals is to employ successfully gene-targeted embryonic stem (ES) cells, in which a transgene has been properly inserted into the predefined locus by homologous recombination. These genetically modified (GM) ES cells can be used for production of chimeric mice by aggregation with normal embryonic cells. Through mating of these resulting chimeric mice with other normal mice, the researcher can obtain Tg animals in which the transgenes have been inserted into the desired locus. Unfortunately, such process requires labor, time consuming, and cost a lot of expenses.
RMCE strategy
To simplify these complex processes, we newly developed a system called “Pronuclear Injection-based Targeted Transgenesis (PITT)”. This system is just like the in vivo version of Cre/loxP-based recombinase-mediated cassette exchange (RMCE). Once “seed mice” that harbor landing pads in their desired locus are established, it is possible to replace the old cassette by a new one containing DNA of interest (DOI) using Cre recombinase. This can easily be performed by microinjecting DNA (comprised by Cre expression plasmid together with the plasmid carrying a new DOI) into the pronuclei of the seed mice-derived zygotes. Using this PITT, we have produced several types of Tg mouse lines. Notably, in most cases we found highly expected and reproducible expression pattern of the introduced transgenes in those mice. The typical example for this case is shown in the attached figure. In this photograph, there are several mice expressing each different types of fluorescent proteins. Remarkably, each fluorescence is very bright and expressed through an entire body, which means unbiased expression of transgenes in vivo. These mice are therefore suitable for transplantation experiments. Some of these fluorescent mice are available from the RIKEN BRC.
In the initial step of PITT development, the efficiency of RMCE in the seed mice-derived zygotes was 4 to 5%. Germline transmission efficiency of PITT founder (F0) mice is up to ~90%. We have also demonstrated that this PITT system is useful for efficient knockdown of a target gene. Therefore, we believe that our PITT system is a “next generation” Tg mouse production system that guarantees unbiased transgene expression (Ohtsuka et al. 2012）.
improved PITT (i-PITT)
As mentioned above, the PITT enables gene replacement at zygote stage. However, the low efficiency of gene replacement at that stage and use of mice with mixed genetic background have been one of our major obstacles to be dissolved. To improve the RMCE efficiency at zygote stage, we used Cre mRNA instead of using a Cre expression plasmid for zygote microinjection. As a result, its efficiency increased up to ~20% (Ohtsuka et al. 2013).
Usage of mouse strain with pure genetic background is important in doing experiments using mice, since it is well known that gene expression is deeply affected by the differences in the genetic background. We therefore made new seed mice (termed “TOKMO-3”) with C57BL/6N genetic background, whose background is now being recognized as standard one for mouse molecular genetic analysis. These resulting new mice have attP site (recognized by phiC31 integrase) and FRT sites (recognized by FLP as FLP/FRT site-specific recombination system) together with mutated loxP sites in their Rosa26 locus. We found that simultaneous PI of multiple recombination-related mRNA such as Cre and PhiC31 mRNA together with donor DNA increased the insertion efficiency of up to 20~30%, when compared with PI of single mRNA alone. We thus named this improved system “improved PITT (i-PITT)”. Furthermore, single PI of multiple donor DNAs (up to three donor vectors) together with two mRNAs coding for recombinase/integrase resulted in production of various types of Tg individuals (namely, Tg lines expressing red, green or blue fluorescence) (Ohtsuka et al. 2015).
Genome-editing via Oviductal Nucleic Acids Delivery (GONAD)
As mentioned previously, PI of nucleic acids at zygote stage is now widely used for creation of GM mice. It requires expensive micromanipulator system and professional persons to operate this apparatus. Furthermore, it also requires ex vivo handling of zygotes such as isolation of zygotes from oviducts, PI towards zygotes, cultivation of the injected zygotes for a short period of time, and return of the injected embryos to the recipient female’s reproductive tracts to deliver pups. To omit these laborious steps, we have intended to introduce nucleic acids into preimplantation embryos floating within an oviduct in vivo. We found that it is possible when a small amount of nucleic acids (including genome-editing tools) is instilled by a glass micropipette under observation using a dissecting microscope and subsequently the entire oviduct is electroporated in vivo using tweezer-type electrodes (see the attached figure). We called this “Genome-editing via Oviductal Nucleic Acids Delivery (GONAD)” (Takahashi et al. 2015). After GONAD procedure, the electroporated females were allowed to deliver pups. Inspection of these pups demonstrated that some had indel mutations in the target locus. Recently, we observed that the mutations caused by this technology can be transmitted to the next generation (unpublished). As mentioned above, this GONAD does not require ex vivo handling of zygotes for creating GM mice. Therefore, this technology will be used for another experimental animals such a rats, and also for various types of animals that are thought to have the difficulty in doing ex vivo handling of embryos, which may include pigs and calves.
GONAD method
improved GONAD (i-GONAD)
After that, we succeeded in increasing the efficiency of the GONAD method by optimizing the time of the experiment and the genome editing reagents, and now call it as "improved GONAD (i-GONAD) method". Various types of genetically modified mice (knockout mice in which specific genes have been disrupted, mice in which minute changes have been introduced into the genes, knock-in mice in which foreign genes have been inserted, etc.) can be produced using the i-GONAD method.
Efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR)
Developing a technique to make the DOI knock-in (KI) into the endogenous target locus is thought to be one of the key subjects in the field of CRISPR/Cas9-based genome editing. We have recently developed a novel system called "Easi-CRISPR" that allows efficient KI of the DOI using a CRISPR/Cas9-based genome editing system (Miura et al. Sci Rep 2015, Nat Protoc 2018, Quadros et al. Genome Biol 2017). The previous method to perform KI is based on the use of single-stranded oligonucleotide (ssODN) that generally spans ~200 bp as a donor. In almost cases, this ssODN contains sequences that are homologous to the target region at its both sides. It is well known that the size of donor DNA is one of the critical factors for efficient KI. We thought that it may be possible if the traditional reverse transcriptase-based amplification of cDNA is applied to synthesize a single-stranded DNA (ssDNA). As a result, we found out that it worked well and called it “in vitro Transcription and Reverse Transcription (ivTRT)” (Miura et al. Sci Rep 2015). Using this in vitro synthesized ssDNA, we achieved the KI efficiency as high as 83%, which is much higher than the previous efficiency (~10%), and thus named the system "Easi-CRISPR". This technology appears to be powerful not only for production of KI mice, but also for basic research in various fields of studies including gene therapy.