Characterizing a lethal CAG-ACE2 transgenic mouse model for SARS-CoV-2 infection using Cas9-enhanced nanopore sequencing.

The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the common animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology.

A potent, broadly neutralizing human monoclonal antibody that efficiently protects hACE2-transgenic mice from infection with the Wuhan, BA.5, and XBB.1.5 SARS-CoV-2 variants.

The COVID-19 pandemic has uncovered the high genetic variability of the SARS-CoV-2 virus and its ability to evade the immune responses that were induced by earlier viral variants. Only a few monoclonal antibodies that have been reported to date are capable of neutralizing a broad spectrum of SARS-CoV-2 variants. Here, we report the isolation of a new broadly neutralizing human monoclonal antibody, iC1.

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene.

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra.

Structural variants in the Epb41l4a locus: TAD disruption and Nrep gene misregulation as hypothetical drivers of neurodevelopmental outcomes.

Structural variations are a pervasive feature of human genomes, and there is growing recognition of their role in disease development through their impact on spatial chromatin architecture. This understanding has led us to investigate the clinical significance of CNVs in noncoding regions that influence TAD structures. In this study, we focused on the Epb41l4a locus, which contains a highly conserved TAD boundary present in both human chromosome 5 and mouse chromosome 18, and its association with neurodevelopmental phenotypes.

The human EF1a promoter does not provide expression of the transgene in mice.

In this work, we set out to create mice susceptible to the SARS-CoV-2 coronavirus. To ensure the ubiquitous expression of the human ACE2 gene we used the human EF1a promoter. Using pronuclear microinjection of the transgene construct, we obtained six founders with the insertion of the EF1a-hACE2 transgene, from which four independent mouse lines were established.

Experimental Study of Antitumor Activity of Pefagtal Addressed to αvβ3 Integrins.

We studied the effect of a new targeted drug Pefagtal that represents a conjugate in which the MS2 phage filled with a substance toxic to cells (thallium salts) is covalently linked to peptides containing the RGD motif. The antitumor and pronounced antimetastatic effects of Pefagtal were demonstrated on transplanted mouse tumors differing in histological type and status of metastasis: Krebs-2 ascites adenocarcinoma of the mammary gland, Lewis lung adenocarcinoma, hepatoma-29, and lung adenocarcinoma. It is assumed that the RGD motif mediates primary binding of the construct to αvβ3 and αvβ5 integrins that are predominantly overexpressed in the endothelial cells of tumor blood vessels and in tumor and metastatic cells.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration.

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), β-casein (Csn2), whey acidic protein (WAP), or β-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization.

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection.

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals.

Pannexin 1 Transgenic Mice: Human Diseases and Sleep-Wake Function Revision.

In humans and other vertebrates pannexin protein family was discovered by homology to invertebrate gap junction proteins. Several biological functions were attributed to three vertebrate pannexins members. Six clinically significant independent variants of the gene lead to human infertility and oocyte development defects, and the Arg217His variant was associated with pronounced symptoms of primary ovarian failure, severe intellectual disability, sensorineural hearing loss, and kyphosis.

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes.

Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results.

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote.

Mechanisms that ensure repair of double-strand DNA breaks (DSBs) are instrumental in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found as a concatemer comprising multiple co-integrated transgene copies. Here, we investigated the contribution of various DSB repair pathways to the concatemer formation.

Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology.

Background: Copy Number Variation (CNV) of the human CNTN6 gene (encoding the contactin-6 protein), caused by deletions or duplications, is responsible for severe neurodevelopmental impairments, often in combination with facial dysmorphias. Conversely, deleterious point mutations of this gene do not show any clinical phenotypes. The aim of this study is to generate mice carrying large deletions, duplications and inversions involving the Cntn6 gene as a new experimental model to study CNV of the human CNTN6 locus.

Unexpected phenotypic effects of a transgene integration causing a knockout of the endogenous Contactin-5 gene in mice.

Contactins (Cntn1-6) are a family of neuronal membrane proteins expressed in the brain. They are required for establishing cell-to-cell contacts between neurons and for the growth and maturation of the axons. In humans, structural genomic variations in the Contactin genes are implicated in neurodevelopmental disorders.

[RELEVANT PRINCIPLES IN THE DIAGNOSIS, TREATMENT, AND PREVENTION OF TOXOPLASMOSIS DURING PREGNANCY].

A retrospective survey of the prevalence of TORCH infections among pregnant women was performed in the perinatal center, M. A. Tverye Military Sanitary Unit Nine (Perm), in June 2010 to December 2013.

[Ethological competence of Perm Territory dwellers in the assurance of personal infection safety].

The authors attempted to analyze preventive measures against infectious and parasitic diseases, which were used domestically by the Perm Territory population, their conjugacy with the stereotypes of attitude towards domestic animals, as well as behavioral features of compliance andcompetence in the assurance of infection safety. The found gaps in the assurance of personal infection safety (drinking unboiled water, unprotected sex, disregard of helminth prevention in domestic animals, and unwillingness to go in for sports) are coherent with the epidemiological situation in the Perm Territory and to our clinical and laboratory study of the patients of the Perm Territory Children's Clinical Hospital in 2011. Enzyme immunoassay (EIA) was used to examine 10075 patients for helminths and protozoa; parasitic diseases were detected in 2047 (20.

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR.

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed.

Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.

Dynamics of recombinant hG-CSF in transgenic goat: preliminary study in the founder during hormonally induced lactation.

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.

A 3,387 bp 5'-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice.

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively.

Pronuclear embryo yield in Canindé and Saanen goats for DNA microinjection.

The objective of this study was to examine the effect of donor breed on pronuclear-stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen-cloprostenol treatment. Forty-eight hours before the sponge removal, superovulation was induced with a total administration of 4.

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil.

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100 microg GnRH 36 h after sponge removal.