Genetically modified mice as a tool for the study of human diseases.

Modeling a human disease is an essential part of biomedical research. The recent advances in the field of molecular genetics made it possible to obtain genetically modified animals for the study of various diseases. Not only monogenic disorders but also chromosomal and multifactorial disorders can be mimicked in lab animals due to genetic modification. Even human infectious diseases can be studied in genetically modified animals. An animal model of a disease enables the tracking of its pathogenesis and, more importantly, to test new therapies. In the first part of this paper, we review the most common DNA modification technologies and provide key ideas on specific technology choices according to the task at hand. In the second part, we focus on the application of genetically modified mice in studying human diseases.

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

AAV infection of bovine embryos: Novel, simple and effective tool for genome editing.

Adeno-associated viruses (AAV) are widely used in the field of genetically modified organism production. In this work, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications: we have used recombinant AAV to produce GFP-positive bovine embryos. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos. AAV9 serotype demonstrated minimal effectiveness (38,10%) as the genetic material transfer tool. Four other serotypes of AAVs (AAV1, AAV2, AAV6 and AAV-DJ) showed very close transduction efficiency (52,94-58,33%). CD209 is a C-type lectin receptor which is presented on the surface of macrophages and dendritic cells. CD209 recognizes a broad range of pathogens in a rather nonspecific manner. Production of CD209 knock-out is relevant for better understanding of infection mechanisms. Potentially, production of such knock-out may enable animals to become resistant to various infections. We have analyzed DNA samples from 22 blastocysts obtained after in vitro culture of zygotes subjected to recombinant AAV action. We have detected that 3 of 22 analyzed blastocysts contained mosaic CD209 frameshifts. Therefore, we have demonstrated proof of principle that application of AAV as a genome editing tool is an effective method for obtaining genetically modified cattle embryos.

Controlled hatching at the prescribed site using femtosecond laser for zona pellucida drilling at the early blastocyst stage.

PURPOSE: To study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability. METHODS: Mouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5-8.5 µm. Additional longitudinal incisions (5-7-µm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated. RESULTS: Femtosecond laser-assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening's small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3-94.7% vs. 83.3-85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups. CONCLUSIONS: Blastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.

Application of femtosecond laser microsurgery in assisted reproductive technologies for preimplantation embryo tagging.

Femtosecond laser pulses were applied for precise alphanumeric code engraving on the zona pellucida (ZP) of mouse zygotes for individual embryo marking and their identification. The optimal range of laser pulse energies required for safe ZP microsurgery has been determined. ZP was marked with codes in three different planes to simplify the process of embryo identification. No decrease in developmental rates and no morphological changes of embryos post laser-assisted engraving have been observed. ZP thickness of embryos post laser-assisted code engraving has been shown to differ significantly from that of control group embryos at the hatching stage. Due to moderate ZP thinning as compared to its initial width at 0.5 dpc (days post coitum), readability of the code degrades slightly and it still remains recognizable even at hatching stage. Our results demonstrate that application of femtosecond laser radiation could be an effective approach for noninvasive direct embryo tagging, enabling embryo identification for the whole period of preimplantation development.

Femtosecond laser is effective tool for zona pellucida engraving and tagging of preimplantation mammalian embryos.

PURPOSE: Our purpose was to study whether application of femtosecond laser pulses for alphanumeric code marking in the volume of zona pellucida (ZP) could be effective and reliable approach for direct tagging of preimplantation embryos. METHODS: Femtosecond laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz, pulse energy of 20 nJ) were applied for precise alphanumeric code engraving on the ZP of mouse embryos at the zygote stage for individual embryo marking and their accurate identification. Embryo quality assessment every 24 h post laser-assisted marking as well as immunofluorescence staining (for ICM/TE cell number ratio calculation) were performed. RESULTS: Initial experiments have started with embryo marking in a single equatorial plane. The codes engraved could be clearly recognized until the thinning of the ZP prior to hatching. Since embryo may change its orientation during the ART cycle, multi-plane code engraving seems to be more practical for simplifying the process of code searching and embryo identification. We have marked the ZP in three planes, and no decrease in developmental rates as well as no morphological changes of embryos post laser-assisted engraving have been observed as compared to control group embryos. CONCLUSIONS: Our results demonstrate the suitability of femtosecond laser as a novel tool for noninvasive embryo tagging, enabling embryo identification from day 0.5 post coitum to at least early blastocyst stage. Thus, the versatility and the potential use of femtosecond lasers in the field of developmental biology and assisted reproduction have been shown.