Cell-marking techniques for cell lineage tracing

A zygote, the only totipotent cell of the developing organism, transforms into a complex, multicellular entity with billions (for humans) of highly specialized cells and tissues. Most adult tissues are maintained by a combination of highly dynamic processes of senescence, apoptosis and rejuvenation with new cells constantly arising from dispersed depots of stem cells. Studying individual cell fates and their intertwined relations thus aids in understanding ontogenetic development as well as pathogenic processes in the body. Direct observations of developing embryos uncovered the fate of single blastomeres of ascidia and nematode. In both cases, research benefited from the simplicity of these objects, because, in ascidia, each blastomere has unique signatures naturally, and, in nematode, transparency of a worm’s 959-cell body allows every cell to be traced through development individually. In most cases, however, studying cellular lineages and identification of stem cells’ subpopulations are a true challenge for investigators. To trace the cell’s fate, novel methods were invented that introduce special tags into cells, the tags that would be inherited during cell divisions. Every descendent of a marked cell bears the same tag and can easily be distinguished from unrelated cellular neighbors. This review focuses on modern methods for cell tracing with dyes and genetic constructs encoding protein reporters that mark cell lineages. Special focus is on genome-integrated tags (genetic labeling), such as viral and cellular barcoding. One chapter of the review describes novel advancements in the field of CRISPR/Cas9-based cellular barcoding

Creation of transgenic mice susceptible to coronaviruses: a platform for studying viral pathogenesis and testing vaccines

Over the past 20 years, coronaviruses have caused three epidemics: SARS-CoV, MERS-CoV, and SARS-CoV2, with the first two having a very high lethality of about 10 and 26 %, respectively. The last outbreak of coronavirus infection caused by SARS-CoV2 in 2019 in China has swept the entire planet and is still spreading. The source of these viruses in humans are animals: bats, Himalayan civets, and camels. The genomes of MERS-CoV, SARS-CoV and SARS-CoV2 are highly similar. It has been established that coronavirus infection (SARS-CoV and SARS-CoV2) occurs through the viral protein S interaction with the lung epithelium – angiotensin-converting enzyme receptor 2 (ACE2) – due to which the virus enters the cells. The most attractive model for studying the development of these diseases is a laboratory mouse, which, however, is resistant to coronavirus infection. The resistance is explained by the difference in the amino acid composition of mouse Ace2 and human ACE2 proteins. Therefore, to create mice susceptible to SARS-CoV and SARS-CoV2 coronaviruses, the human ACE2 gene is transferred into their genome. The exogenous DNA of the constructs is inserted into the recipient genome randomly and with a varying number of copies. Based on this technology, lines of transgenic mice susceptible to intranasal coronavirus infection have been created. In addition, the use of the technology of targeted genome modification using CRISPR/Cas9 made it possible to create lines of transgenic animals with the insertion of the human ACE2 gene under the control of the endogenous murine Ace2 gene promoter. This “humanization” of the Ace2 gene makes it possible to obtain animals susceptible to infection with coronaviruses. Thus, transgenic animals that simulate coronavirus infections and are potential platforms for testing vaccines have now been created

Reorganisation of chromatin during erythroid differentiation

A totipotent zygote has unlimited potential for differentiation into all cell types found in an adult organism. During ontogenesis proliferating and maturing cells gradually lose their differentiation potential, limiting the spectrum of possible developmental transitions to a specific cell type. Following the initiation of the developmental program cells acquire specific morphological and functional properties. Deciphering the mechanisms that coordinate shifts in gene expression revealed a critical role of three-dimensional chromatin structure in the regulation of gene activity during lineage commitment. Several levels of DNA packaging have been recently identified using chromosome conformation capture based techniques such a Hi-C. It is now clear that chromatin regions with high transcriptional activity assemble into Mb-scale compartments in the nuclear space, distinct from transcriptionally silent regions. More locally chromatin is organized into topological domains, serving as functionally insulated units with cell type – specific regulatory loop interactions. However, molecular mechanisms establishing and maintaining such 3D organization are yet to be investigated. Recent focus on studying chromatin reorganization accompanying cell cycle progression and cellular differentiation partially explained some aspects of 3D genome folding. Throughout erythropoiesis cells undergo a dramatic reorganization of the chromatin landscape leading to global nuclear condensation and transcriptional silencing, followed by nuclear extrusion at the final stage of mammalian erythropoiesis. Drastic changes of genome architecture and function accompanying erythroid differentiation seem to be an informative model for studying the ways of how genome organization and dynamic gene activity are connected. Here we summarize current views on the role of global rearrangement of 3D chromatin structure in erythroid differentiation

CRISPR/Cas9, a universal tool for genomic engineering

The CRISPR/Cas9 system was initially described as an element of archeal and bacterial immunity, but gained much attention recently for its outstanding ability to be programmed to target any genomic loci through a short 20-nucleotide sgRNA region. Here we review some modern applications of the CRISPR/Cas9 system. First, we describe the basic mechanism of the CRISPR/Cas9 DNA recognition and binding, focusing in particular on its off-target activity. The CRISPR/Cas9 off-target activity refers to a non-specific recognition of genomic sites that have partial homology with sgRNA, occasionally resulting in unwanted mutations throughout the genome. We also note some recent improvements for enhancing Cas9 specificity or adding new functions to the system. Since Cas9-related hype is mostly driven by its remarkable potential for gene therapy and genome engineering, the latest CRISPR/Cas9 applications in these areas are also covered in our review. For instance, the CRISPR/Cas9 was recently used to control HIV infection and to repair genetic abnormalities, such as Duchenne muscular dystrophy or retinitis pigmentosa, both in cell cultures and rodent models. A programmable nature of CRISPR/Cas9 facilitates the creation of transgenic organisms through sitespecific gene mutations, knock-ins or large chromosomal rearrangements (deletions, inversions and duplications). CRISPR/Cas9 proved to be especially useful in pronuclear microinjections of farm animals as well, having strong impact on biotechnology. In addition, we review Cas9-augmented genetic screens that allow an unbiased search for new genes and pathways involved in a plethora of biological aspects, owing to Cas9 efficiency and versatility. Finally, we argue that gene drivers based on CRISPR/ Cas9 represent a powerful tool to modify ecosystems in the nearest future

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. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression

Efficient chimeric mouse production using a novel embryonic stem cell line

Embryonic stem cells are commonly used for generation of transgenic mice. Embryonic stem cells could participate in the development of chimeric animals after injection into a blastocyst. Injection of genetically modified embryonic stem cells could lead to germ line transmission of a transgene or genomic modification in chimeric mice. Such founders are used to produce transgenic lines of mice. There are several projects dedicated to production of knock-out mouse lines (KOMP Repository, EUCOMM, Lexicon Genetics). Never-theless, there is a need for complex genome modifications, such as large deletions, reporter genes insertion into the 3’ gene regulatory sequence, or site-specific modifications of the genome. To do that, researchers need an embryonic stem cell line that is able to participate in chimeric animal formation even after prolonged culture in vitro. Several lines of mouse embryonic stem cells were produced in the Laboratory of Developmental Genetics of the Institute of Cytology and Genetics SB RAS. We tested DGES1 cell line (2n = 40, XY) (129S2/SvPasCrl genetic background) for chimeric mice production at the Center for Genetic Resources of Laboratory Animals at ICG SB RAS. Embryonic stem cells were injected into 136 blastocysts (B6D2F1 genetic background), which were transplanted into CD-1 mice. Among 66 progeny, 15 were chimeric, 4 of which were more than 80 % chimeric judged by coat color. All chimeras were males without developmental abnormalities. 10 of 15 males were fertile. Microsatellite analysis of the progeny of chimeric mice revealed embryonic stem cell line DGES1 contribution to the gamete formation. Thus, a novel DGES1 embryonic stem cell line could be efficiently used for transgenic mouse production using B6D2F1 blastocysts and CD-1 recipients

Morphophysiological alterations caused by insertional mutagenesis of contactin 5 (Cntn5) gene in transgenic mice

Transgenesis has become a routine for modern biological studies. The most popular method for producing transgenic animals–pronuclear microinjection–frequently leads to host gene disruption due to a random transgene integration. In this paper, we report our analysis of morphophysiological parameters of the transgenic mouse line GM9, in which a transgene designed for milk-specific expression of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was integrated into the intron of the Contactin 5 gene (Cntn5). We studied Cntn5 expression with RT-PCR and discovered that its expression in the brain, the primary organ of Cntn5 activity, was unperturbed. However, transgenic animals had less Cntn5 transcripts in other tissues such as the kidney and heart. In addition, we observed a decreased amount of splice variants of Cntn5 exons that flank the transgene integration site. These data suggest that the transgene integration event might affect proper Cntn5 splicing in some tissues. Publications exist that imply that some polymorphisms in the Cntn5 gene are associated with obesity and arterial hypertension in humans. We evaluated core parameters of lipid metabolism and heart activity in mice homozygous and heterozygous for Cntn5 mutation using wild- type animals as control. Our results uncovered that homozygous mutant mice have lower body weight than controls and that it is caused by slower accumulation of fat tissue. Cntn5 mutants also exhibit abnormalities in blood circulation: homozygous Cntn5 mutants are characterized by a higher blood pressure and heart beat rate, as well as faster blood flow in the tail vessels. Heterozygous animals showed intermediate results for all of these parameters

3C-BASED METHODS FOR 3D GENOME ORGANIZATION ANALYSIS

The 3D organization of an eukaryotic genome plays an important role in nuclear gene expression. Until recently, studies of this organization were limited by light microscopy and electron microscopy methods. The development of chromosome conformation capture (3С) methods allowed studying genome-wide chromosomal contacts by using only molecular methods. Nowadays, numerous 3C-based methods have been developed to reconstruct the 3D organization of eukaryotic genomes

INVESTIGATION OF THE SPATIAL GENOME ORGANIZATION OF MOUSE SPERM AND FIBROBLASTS BY THE Hi-C METHOD

The spatial organization of an eukaryotic genome plays an important role in the control of nuclear gene expression. The new Hi-C method allows investigation of the three-dimensional architecture of whole genomes. It has not been applied to study of the spatial configuration of a germ cell genome hitherto. Here we describe a protocol for production and quality control of Hi-C libraries from fibroblasts and sperm cells. Our results demonstrate that the Hi-C method can be used for studying the spatial organization of the densely packed sperm genome