Optimal Number of Embryos for Transplantation in Obtaining Genetic-Modified Mice and Goats

Silaeva, Yulia Yu.; KIRIKOVICH, Y. K.; Skuratovskaya, L. N.; Deikin, A. V.

doi:10.1134/s106236041806005x

Cited by 10 publications

(6 citation statements)

References 24 publications

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“…For blastocyst assay, the zygotes were transferred into 35 mm Petri dishes and cultured in 50–60 μL droplets of KSOM medium (MTI-GlobalStem) under mineral oil at 37°C and 5% CO2 for 3 days till the blastocyst stage. To produce genome-modified mice, injected eggs were incubated overnight, and viable two-cell-stage embryos were implanted into pseudopregnant foster dams ( Silaeva et al, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

et al. 2023

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The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.

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Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

et al. 2023

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“…The transgene DNA construct was introduced by pronuclear microinjections as described earlier (Zvezdova et al 2010 ). Surviving zygotes were cultivated in M16 medium at 37 °C and 6% CO 2 for 24 h and then transplanted into the oviducts of foster mothers at the 2-cell stage as previously described (Silaeva et al 2018 ).…”

Section: Methodsmentioning

confidence: 99%

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis

et al. 2021

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Graphic abstract The current coronavirus disease (COVID-19) pandemic remains one of the most serious public health problems. Increasing evidence shows that infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a very complex and multifaceted disease that requires detailed study. Nevertheless, experimental research on COVID-19 remains challenging due to the lack of appropriate animal models. Herein, we report novel humanized mice with Cre-dependent expression of hACE2, the main entry receptor of SARS-CoV-2. These mice carry hACE2 and GFP transgenes floxed by the STOP cassette, allowing them to be used as breeders for the creation of animals with tissue-specific coexpression of hACE2 and GFP. Moreover, inducible expression of hACE2 makes this line biosafe, whereas coexpression with GFP simplifies the detection of transgene-expressing cells. In our study, we tested our line by crossing with Ubi-Cre mice, characterized by tamoxifen-dependent ubiquitous activation of Cre recombinase. After tamoxifen administration, the copy number of the STOP cassette was decreased, and the offspring expressed hACE2 and GFP , confirming the efficiency of our system. We believe that our model can be a useful tool for studying COVID-19 pathogenesis because the selective expression of hACE2 can shed light on the roles of different tissues in SARS-CoV-2-associated complications. Obviously, it can also be used for preclinical trials of antiviral drugs and new vaccines.

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“…The issues of modelling processes occurring in bioobjects under the influence of them by a laser energy source have been studied by domestic and foreign scientists. However, they form the basis for the study of applied problems of bioeconomics of laser division of early elite embryos of farm animals and the subsequent use of viable separated parts of the embryo at artificial insemination stations (Csillag et al, 1988;Silaeva et al, 2018).…”

Section: Effectiveness Of Example Bioeconomics Tasksmentioning

confidence: 99%

The Quality Function in Determining the Effectiveness of Example Bioeconomics Tasks

Levkin¹,

Abuselidze

BEREZHNA³

et al. 2022

Rural Sustainability Research

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The aim of the publication is to develop theoretical and methodological foundations and practical recommendations for determining the efficiency of biotechnological processes with the subsequent justification of technical parameters to ensure a high level of viability of bio-objects. In the course of the conducted scientific research, the criteria of quality function were defined and formalised, in accordance with which the conditions of biotechnological processes of laser division of embryo were formulated as a single case in the realisation of the economic mechanism of biotechnology. This makes it possible to ensure a high level of cattle (cattle) productivity, reduce the time for the reproduction of livestock and reduce the costs of movement, trade and transportation of cattle. The quality function of the biotechnological process of laser division of embryo is formulated on the basis of criterion of non-exceeding of the temperature field in cells of blastomeres and the predetermined acceptable value that allowed one to take into account the main parameters and to propose the criterion of biotechnological process optimisation. As a promising task for the development of the bioeconomy as an economic mechanism in implementing biotechnologies, the necessity of determining the quality function by formalising the integral indicator of efficiency has been established, This enables one to ensure a high level of commercialisation of biotechnologies, to increase the productivity of agricultural animals, and to suggest alternative methods for the quick renewal of livestock.

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Optimal Number of Embryos for Transplantation in Obtaining Genetic-Modified Mice and Goats

Cited by 10 publications

References 24 publications

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis

The Quality Function in Determining the Effectiveness of Example Bioeconomics Tasks

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