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. Analysis of human data from the DECIPHER database indicates that CNVs within this locus, including both deletions and duplications, are often observed alongside neurological abnormalities, such as dyslexia and intellectual disability, although there is not enough evidence of a direct correlation or causative relationship. To investigate these possible associations, we generated mouse models with deletion and inversion mutations at this locus and carried out RNA-seq analysis to elucidate gene expression changes. We found that modifications in the Epb41l4a TAD boundary led to dysregulation of the Nrep gene, which plays a crucial role in nervous system development. These findings underscore the potential pathogenicity of these CNVs and highlight the crucial role of spatial genome architecture in gene expression regulation.

Knockout of the Tnfa Gene Decreases Influenza Virus-Induced Histological Reactions in Laboratory Mice.

Tumor necrosis factor alpha (TNF-α) is a cytokine that is responsible for many processes associated with immune response and inflammation. It is involved in the development of an antiviral response to many virus infections. This factor was shown to be activated in influenza A virus infection, which enhances production of other cytokines. The overexpression of these cytokines can lead to a cytokine storm. To study the role of TNF-α in the development of pathologies associated with viral infection, we generated a Tnfa knockout mouse strain. We demonstrated that these mice were characterized by a significant increase in the number of viral genomes compared to that in the parental strain, but the amount of live virus did not differ. A histopathology of the lungs in the genetically modified animals was significantly lower in terms of interalveolar septal infiltration. The generated model may be used to further study pathological processes in viral infections.

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. Unfortunately, only one line had low levels of hACE2 expression in some organs. In addition, we did not detect the hACE2 protein in primary lung fibroblasts from any of the transgenic lines. Bisulfite sequencing analysis revealed that the EF1a promoter was hypermethylated in the genomes of transgenic animals. Extensive analysis of published works about transgenic animals indicated that EF1a transgenic constructs are frequently inactive. Thus, our case cautions against using the EF1a promoter to generate transgenic animals, as it is prone to epigenetic silencing.

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 PANX1 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. At the same time, only mild phenotypes were observed in Panx1 knockout mice. In addition, a passenger mutation was identified in a popular line of Panx1 knockout mice, questioning even those effects. Using CRISPR/Cas9, we created a new line of Panx1 knockout mice and a new line of mice with the clinically significant Panx1 substitution (Arg217His). In both cases, we observed no significant changes in mouse size, weight, or fertility. In addition, we attempted to reproduce a previous study on sleep/wake and locomotor activity functions in Panx1 knockout mice and found that previously reported effects were probably not caused by the Panx1 knockout itself. We consider that the pathological role of Arg217His substitution in Panx1, and some Panx1 functions in general calls for a re-evaluation.

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. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.

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. We injected mouse zygotes with a pool of linear DNA molecules carrying unique barcodes at both ends and obtained 10 transgenic embryos with 1-300 transgene copies. Sequencing the barcodes allowed us to assign relative positions to the copies in concatemers and detect recombination events that occurred during integration. Cumulative analysis of approximately 1,000 integrated copies reveals that over 80% of them underwent recombination when their linear ends were processed by synthesis-dependent strand annealing (SDSA) or double-strand break repair (DSBR). We also observed evidence of double Holliday junction (dHJ) formation and crossing over during the concatemer formations. Sequencing indels at the junctions between copies shows that at least 10% of DNA molecules introduced into the zygotes are ligated by non-homologous end joining (NHEJ). Our barcoding approach, verified with Pacific Biosciences Single Molecule Real-Time (SMRT) long-range sequencing, documents high activity of homologous recombination after DNA microinjection.

Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice.

In a previous study using one-step CRISPR/Cas9 genome editing in mouse zygotes, we created five founders carrying a 1,137 kb deletion and two founders carrying the same deletion, plus a 2,274 kb duplication involving the Cntn6 gene (encoding contactin-6). Using these mice, the present study had the following aims: (i) to establish stage of origin of these rearrangements; (ii) to determine the fate of the deleted DNA fragments; and (iii) to estimate the scale of unpredicted DNA changes accompanying the rearrangements. The present study demonstrated that all targeted deletions and duplications occurred at the one-cell stage and more often in one pronucleus only. FISH analysis revealed that there were no traces of the deleted DNA fragments either within chromosome 6 or on other chromosomes. These data were consistent with the Southern blot analysis showing that chromosomes with deletion often had close to expected sizes of removed DNA fragments. High-throughput DNA sequencing of two homozygotes for duplication demonstrated that there were no unexpected significant or scale DNA changes either at the gRNA and joint sites or other genome sites. Thus, our data suggested that CRISPR/Cas9 technology could generate megabase-sized deletions and duplications in mouse gametes at a reasonably specific level.

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. In addition, population genetic studies associate Contactins loci with obesity and hypertension. Cntn5 knockout mice were first described in 2003, but showed no gross physiological or behavioral abnormalities (just minor auditory defects). We report a novel Cntn5 knockout mouse line generated by a random transgene integration as an outcome of pronuclear microinjection. Investigation of the transgene integration site revealed that the 6Kbp transgene construct coding for the human granulocyte-macrophage colony-stimulating factor (hGMCSF) replaced 170 Kbp of the Cntn5 gene, including four exons. Reverse transcription PCR analysis of the Cntn5 transcripts in the wild-type and transgenic mouse lines showed that splicing of the transgene leads to a set of chimeric hGMCSF-Cntn5 transcript variants, none of which encode functional Cntn5 protein due to introduction of stop codons. Although Cntn5 knockout animals displayed no abnormalities in behavior, we noted that they were leaner, with less body mass and fat percentage than wild-type animals. Their cardiovascular parameters (heart rate, blood pressure and blood flow speed) were elevated compared to controls. These findings link Cntn5 deficiency to obesity and hypertension.

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. RESULTS: To generate large chromosomal rearrangements on mouse chromosome 6, we applied CRISPR/Cas9 technology in zygotes. Two guide RNAs (gRNAs) (flanking a DNA fragment of 1137 Mb) together with Cas9 mRNA and single-stranded DNA oligonucleotides (ssODN) were microinjected into the cytoplasm of 599 zygotes of F1 (C57BL x CBA) mice, and 256 of them were transplanted into oviducts of CD-1 females. As a result, we observed the birth of 41 viable F0 offspring. Genotyping of these mice was performed by PCR analysis and sequencing of PCR products. Among the 41 F0 offspring, we identified seven mice with deletions, two animals carrying duplications of the gene and four carrying inversions. Interestingly, two F0 offspring had both deletions and duplications. It is important to note that while three of seven deletion carriers showed expected sequences at the new joint sites, in another three, we identified an absence of 1-10 nucleotides at the CRISPR/Cas9 cut sites, and in one animal, 103 bp were missing, presumably due to error-prone non-homologous end joining. In addition, we detected the absence of 5 and 13 nucleotides at these sites in two F0 duplication carriers. Similar sequence changes at CRISPR/Cas9 cut sites were observed at the right and left boundaries of inversions. Thus, megabase-scale deletions, duplications and inversions were identified in 11 F0 offspring among 41 analyzed, i.e., approximately 25% efficiency. All genetically modified F0 offspring were viable and able to transmit these large chromosomal rearrangements to the next generation. CONCLUSIONS: Using CRISPR/Cas9 technology, we created mice carrying megabase-scale deletions, duplications, and inversions involving the full-sized Cntn6 gene. These mice became founders of new mouse lines, which may be more appropriate experimental models of CNV in the human 3p26.3 region than Сntn6 knockout mice.

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. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

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 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.

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. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 µg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.

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. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

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 100µg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

A fim de produzir caprinos transgênicos para o hG-CSF, utilizou-se 24 cabras Saanen adultas e 48 cabras sem raça definida adultas como doadoras e receptoras, respectivamente. As doadoras tiveram o estro sincronizado por esponjas vaginais e foram superovuladas com 200 mg de FSH em doses decrescentes, duas vezes ao dia e iniciando 48 h antes da retirada da esponja. A ovulação foi induzida pela injeção de 100 µg de GnRH às 36 h após a retirada da esponja. As receptoras também receberam um tratamento de sincronização do estro. As doadoras foram cobertas por bodes Saanen férteis e, aproximadamente 72 h após a retirada da esponja, os zigotos foram colhidos cirurgicamente por lavagem dos ovidutos. Os zigotos colhidos foram rapidamente centrifugados para uma melhor visualização dos pró-núcleos. A construção de DNA, contendo o gene do hG-CSF flanqueado pelos genes caprino e bovino da alfas1-caseína, foi injetada em 129 embriões. Os embriões microinjetados (3 a 6 por receptora) foram transferidos para 27 receptoras que responderam ao tratamento. Dez receptoras ficaram gestantes e 12 crias foram produzidas. Um macho transgênico fundador foi identificado no grupo de crias nascidas. Este é o primeiro relato do nascimento de um caprino transgênico na América Latina.

Genes and chromosomes: control of development

Durante a última década houve imenso progresso na pesquisa sobre as bases moleculares da regulação gênica durante o desenvolvimento. Foram identificados grupos de genes funcionando sob controle hierárquico, sistemas de genes conservados ao longo da evolução atuando na transmissão célula a célula de sinais transmembrana e com uma função central na morfogênese foram intensamente estudados e o conceito de redes genômicas regulatórias coordenando a expressão de diversos genes foi introduzido, para citar apenas alguns dos principais avanços. Deve-se notar que os parâmetros tempo e tecido-específicos da expressão gênica são corretamente regulados durante o desenvolvimento apenas no contexto do cromossomo e que são amplamente dependentes da posição do gene no cromossomo ou no núcleo em interfase. Além do mais, a herança epigenética dos estados gênicos através de sucessivas geraçäes celulares foi conduzida exclusivamente ao nível cromossômico em função da memória celular ou cromossômica. A memória ontogenética é uma propriedade inerente do cromossomo e a regulação em cis tem um papel crucial em sua manutenção.

Genes and chromosomes: control of development.

The past decade has witnessed immense progress in research into the molecular basis behind the developmental regulation of genes. Sets of genes functioning under hierarchical control have been identified, evolutionary conserved systems of genes effecting the cell-to-cell transmission of transmembrane signals and assigned a central role in morphogenesis have been intensively studied; the concept of genomic regulatory networks coordinating expression of many genes has been introduced, to mention some of the major breakthroughs. It should be noted that the temporal and tissue-specific parameters of gene expression are correctly regulated in development only in the context of the chromosome and that they are to a great extent dependent on the position of the gene on the chromosome or the interphase nucleus. Moreover epigenetic inheritance of the gene states through successive cell generations has been conducted exclusively at the chromosome level by virtue of cell or chromosome memory. The ontogenetic memory is an inherent property of the chromosome and cis-regulation has a crucial role in its maintenance.

Genetic modification of mammalian genome at chromosome level

The review is concerned with a progress in genetic modification of a mammalian genome in vitro and in vivo at chromosomal level. Recently three new approaches for the chromosome biotechnology have been developed: Using Cre/loxP-system a researcher is able to produce targeted rearrangements of whole chromosomes or their segments or particular genes within the genome, and therefore to modify the set, position and copy number of the endogenous elements of the genome. Mammalian artificial chromosomes (MACs) provide a possibility to introduce into genome relatively large segments of alien chromosome material, either artificially constructed or derived from the genome of different species. Using ES-somatic cell hybrids allows to transfer whole chromosomes or their fragments between different genomes within and between species. Advantages and limitations of these approaches are discussed.