CRISPR/Cas9 Essential Gene Editing in Drosophila.

Since the addition of the CRISPR/Cas9 technology to the genetic engineering toolbox, the problems of low efficiency and off-target effects hamper its widespread use in all fields of life sciences. Furthermore, essential gene knockout usually results in failure and it is often not obvious whether the gene of interest is an essential one. Here, we report on a new strategy to improve the CRISPR/Cas9 genome editing, which is based on the idea that editing efficiency is tightly linked to how essential the gene to be modified is. The more essential the gene, the less the efficiency of the editing and the larger the number of off-targets, due to the survivorship bias. Considering this, we generated deletions of three essential genes in Drosophila: trf2, top2, and mep-1, using fly strains with previous target gene overexpression ("pre-rescued" genetic background).

Study of the Role of Long Noncoding roX RNA in Maintaining of the Dosage Compensation Complex in Drosophila melanogaster.

The proteins MSL1, MSL2, MSL3, MLE, and MOF and noncoding RNAs roX1 and roX2 form the Drosophila dosage compensation complex (DCC), which specifically binds to the X chromosome of males. It is known that noncoding RNA roX are primary component of the DCC in the process of assembly and spreading of the complex among the X chromosome of males. However, the role of this RNA in maintaining the structure of the already assembled complex remains unclear. In this work, we have shown that the full-assembled dosage compensation complex dissociates rather weakly when treated with RNases: the MLE helicase is effectively released from the complex, and the remaining protein components (MSL1, MSL2, and MSL3) undergo partial disassembly and continue to be part of subcomplexes. The results confirm the importance of the noncoding roX2 RNA not only in the processes of initiation of DCC assembly but also at the stage of maintaining the structure of the already assembled complex.

Mutations of Phosphorylation Sites in MSL1 Protein Do Not Affect Dosage Compensation in Drosophila melanogaster.

Proteins MSL1 and MSL2 form the core of the Drosophila dosage compensation complex, which specifically binds to the X chromosome of males. Phosphorylation of certain amino acid residues was previously shown to regulate MSL1 activity. In the present work, transgenic lines of Drosophila expressing mutant variants of the MSL1 protein were obtained, in which amino acids undergoing phosphorylation were replaced. As a result, it was shown that inactivation of phosphorylation sites does not affect the efficiency of specific binding of the dosage compensation complex to the X chromosome of males and its functional activity.

TTK Isoforms Interact with Two Regions of the Mep-1 Protein of Drosophila melanogaster.

The Drosophila TTK protein is involved in the processes of cell differentiation and is represented by two isoforms, TTK69 and TTK88, which have a common N-terminal BTB domain and different C-terminal sequences. Earlier, it was shown that TTK69 represses the activity of enhancers and promoters by recruiting a conserved among higher eukaryotes NURD complex to chromatin. The Mep-1 protein was found in the NURD-complex of Drosophila, and this protein can interact with the C-terminal region of TTK69. In the present study, using the yeast two-hybrid system, we mapped the interacting regions of the TTK and Mep-1 proteins. We identified regions in the unique C-terminal regions of TTK isoforms that can interact simultaneously with two regions of the Mep-1 protein. The results show that, despite the low homology of the C-terminal regions, the TTK isoform retains the ability to interact with two conserved regions of the Mep-1 protein, which suggests the functional significance of this interaction.

CTCF As an Example of DNA-Binding Transcription Factors Containing Clusters of C2H2-Type Zinc Fingers.

In mammals, most of the boundaries of topologically associating domains and all well-studied insulators are rich in binding sites for the CTCF protein. According to existing experimental data, CTCF is a key factor in the organization of the architecture of mammalian chromosomes. A characteristic feature of the CTCF is that the central part of the protein contains a cluster consisting of eleven domains of C2H2-type zinc fingers, five of which specifically bind to a long DNA sequence conserved in most animals. The class of transcription factors that carry a cluster of C2H2-type zinc fingers consisting of five or more domains (C2H2 proteins) is widely represented in all groups of animals. The functions of most C2H2 proteins still remain unknown. This review presents data on the structure and possible functions of these proteins, using the example of the vertebrate CTCF protein and several well- characterized C2H2 proteins in Drosophila and mammals.

Functional Comparison of Short and Long Isoforms of the TRF2 Protein in Drosophila melanogaster.

TRF2 protein (TBP-related factor 2) can substitute for TBP forming alternative transcription initiation complexes on TATA-less promoters, including the promoters of histone H1 and piRNA clusters required for transposon repression. The Drosophilatrf2 gene codes for two isoforms: a "short" and a "long" one, in which the same short TRF2 sequence is preceded by a long N-terminal domain. Here, we demonstrated that the long TFR2 isoform has a greater functional activity than the short isoform by expressing each of them at a reduced rate under the endogenous promoters. Expression of the long isoform alone affects neither the flies' viability nor the sex ratio. Expression of the short isoform alone leads to the phenotype described for the trf2 gene insufficiency and derepression of transposable elements, that is, decreased viability, disturbance of homologous chromosome pairing and segregation, and apparent female-biased sex ratio.

Identification of the ORC Complex Subunits That Can Interact with the ENY2 Protein of Drosophila melanogaster.

The interaction of the Drosophila ENY2 protein with the ORC complex subunits was investigated. It is found that ORC4 and ORC6 subunits directly interact with ENY2.

Study of the Ability of the gypsy Insulator to Stabilize Amplification of the chorion Replication Origin of Drosophila melanogaster.

The role of the gypsy insulator in the replication origin (RO) activity in the presence and absence of one and two copies of this insulator in several genomic sites was studied. Due to the fact that the prepared model system makes it possible to study the activity of this element in a given genomic site, it was shown that the RO stabilization, indeed, is determined by the activity of the insulator rather than by the construct integration site into the genome. The role of the Su(Hw) protein in this process was also studied in detail.

Neonatal Lethality and Inflammatory Phenotype of the New Transgenic Mice with Overexpression of Human Interleukin-6 in Myeloid Cells.

To model human interleukin-6 (hIL-6) associated diseases, unique mice with transgenic overexpression of human IL-6 and reporter fluorescent protein EGFP in cells of macrophage-monocyte lineage were generated using loxP-Cre system. High level of hIL-6 production by macrophages and monocytes, as confirmed in vitro in primary culture of bone marrow-derived macrophages, in vivo resulted in early postnatal death in vivo, presumably, due to the effect of overexpression of hIL-6 on hematopoiesis.

[Optimal Artificial Mini-Introns for Transgenic Expression in the Cells of Mice and Hamsters].

Introns can frequently enhance transgene expression, and sometimes they are absolutely substantial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to be efficiently spliced in CHO cells from hamster ovaries. The proposed strategy can be used to include introns in cDNA, which would elevate the production of recombinant proteins in cell culture, as well as in transgenic animals.

ZAD-Domain Is Essential for Nuclear Localization of Insulator Proteins in Drosophila melanogaster.

Many arthropod zinc-finger transcription factors contain a N-terminal domain called ZAD (Zinc-finger Associated Domain), which consists of four cysteines coordinating a single zinc ion. Dimerization ability has been shown for several ZAD-domains. The functional role of this domain is poorly understood. In this paper, we demonstrate that a point mutation within the ZAD-domain of the Zw5 insulator protein disrupts its nuclear localization without affecting its dimerization ability. The importance of the ZAD-domain for nuclear localization has also been shown for the Pita and Grauzone proteins. Therefore, one of the ZAD-domain functions is control of the nuclear localization of transcription factors.

CTCF and Sgfl1 proteins form alternative complexes with ENY2 proteins.

ENY2 is a multifunctional protein, a component of the SAGA complex (protein complex involved in the regulation of transcription, possessing histone-acetyltransferase and H2B-histone-deubiquitinylating activities [1]) and TREX-2 (complex involved in the mRNA nuclear export [2]). Besides this, the interactions of ENY2 with DNA-binding insulator proteins Su(Hw) and dCTCF have been described as essential for the barrier activity of corresponding insulators [3, 4]. In this study, we described the formation of mutually exclusive complexes of ENY2 with insulator proteins and Sgfl1-a component of the SAGA complex, direct binding partner for ENY2.

[Translation elongation factor EF1α1 interacts with ZAD domains of transcription factors from Drosophila melanogaster].

A large class of arthropod transcription factors is formed by proteins with a characteristic N-terminal Zinc-finger-Associated Domain (ZAD) which contains four cysteine residues that coordinate a zinc ion. The number of putative proteins with ZAD in the Drosophila genome exceeds 90, and the degree of sequence similarity between these domains can be as low as 23%. Efficient binding of ZADs from the proteins Grau, ZIPIC, and Zw5 to the translation elongation factor EF1α1 in nuclear and cytoplasmic extracts has been demonstrated. EF1α1 is probably involved in the regulation of the activity of ZAD-containing transcription factors.

[Induction of transcription through the scs insulator leads to abnormal development of Drosophila melanogaster].

A regulatory element named scs is one of the first insulators discovered in Drosophila, which was found on the boundary of the hsp70 domain. The 993-bp scs insulator contains two promoters at the ends and two polyadenylation signals located in the same orientation in the central part of the insulator. In the Drosophila transgenic lines, induction of a strong transcription through the scs insulator only in the direction that coincides with the direction of the two polyadenylation sites activity results in multiple phenotypic defects of the Drosophila development and embryonic lethality. A similar effect was not observed upon testing of other known Drosophila insulators.

[Recruiting of insulator protein ZIPIC of Drosophila melanogaster to minor binding sites in vivo depends on other DNA-binding transcription factors].

ZIPIC insulator protein of Drosophila has seven zinc finger domains at the C-terminus. Some of this zinc fingers are involved in binding of specific DNA sequence: CAGGGCTG. ZIPIC can interact only in vivo with minor form of this site (substitution of G to T at position 4). Possible explanation is interaction with additional transcription factors can help ZIPIC to bind minor form of consensus. On the other hand ZIPIC can efficiently bind in vitro other minor form of consensus (substitution of C to A at 6 position).

The use of artificial transcription terminators for creating transgenic plants with high-level expression of reporter gene.

In this study, we investigated the possibility of increasing the level of transgene expression using DNA element that can terminate transcription.

The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human ß- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

[Organization of the Drosophila melanogaster SF1 insulator and its role in transcription regulation in transgenic lines].

The SF1 insulator was found to contain a polyadenylation signal, which corresponded to the functional polyadenylation signal in embryonic S2 cells and the transgenic lines of Drosophila and bi-directional promoter that functioned in S2 cells. The studies performed did not confirm the ability of the SF1 insulator to protect expression of reporter gene white from the chromosome position effect in transgenic lines.

Use of transgenic animals in biotechnology: prospects and problems.

During the past two decades, there have been numerous attempts at using animals in order to produce recombinant human proteins and monoclonal antibodies. However, it is only recently that the first two therapeutic agents isolated from the milk of transgenic animals, C1 inhibitor (Ruconest) and antithrombin (ATryn), appeared on the market. This inspires hope that a considerable number of new recombinant proteins created using such technology could become available for practical use in the near future. In this review, the methods applied to produce transgenic animals are described and the advantages and drawbacks related to their use for producing recombinant human proteins and monoclonal antibodies are discussed.