Experimental Study of Antitumor Activity of Pefagtal Addressed to αvß3 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 αvß3 and αvß5 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), ß-casein (Csn2), whey acidic protein (WAP), or ß-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

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.

[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. The survey covered 2060 women: they were all examined for cytomegalovirus, herpes simplex virus, and Toxoplasma. Antibodies to Toxoplasma gondii were detected in 28.68% (591/2060); 98.62% were found to have antibodies to herpes simplex; antibodies to cytomegalovirus were identified in 87.13% (1795/2060). Acute maternal toxoplasmosis was diagnosed by seroconversion or determination of IgM anti-Toxoplasma gondii antibodies, in the presence of a low avidity index and a four-fold increase in antibody titers, by simultaneously studying paired serum samples obtained at a 2-week interval. To confirm fetal infection, amniotic fluid PCR examination should be performed after 18 weeks' gestation. No consensus of opinion as to the principles of treatment for toxoplasmosis in pregnant women makes relevant the long-term results of antibacterial and antiprotozoal treatment cycles varying in duration and intensity. The prevention of acute toxoplasmosis in pregnant women ensures the principle of a mother's personal responsibility for infection safety of a newborn infant, which is informationally provided in health and safety fundamentals course and pregravid preparation schools.

[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 (20.3%) persons. The diagnostic titer of antibodies to Toxocara antigens was revealed in 677 (11.8%) children of 5700 patients examined for toxocariasis; that for Opisthorchis antigens was in 595 (37.7%) of 1578 examined for Opisthorchis infestation. The diagnostic titers for echinococcosis was found in 9 (0.75%) of 1198 patients; later on the diagnosis of hydatid disease was verified by epidemiological, clinical, and laboratory studies. Despite the inadequate informative value of EIA for the diagnosis of giardiasis, high antibody titers to Lamblia antigens were detected in 766 (47.9%) of 1599 children.

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 1.9 to 14 µg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 µg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

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 4.4 mg/kg bodyweight NIH-FSH-P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 µg of GnRH and they were hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.

[Mosaic expression of the lacZ reporter-gene under control of 5'-regulatory sequences of the alpha-S1-casein gene in transgenic mice].

Phenomenon of mosaic expression at cellular level is widely presented in tissues and organs of transgenic animals. The communication is concerned a study of the mosaics in transgenic mice carrying the lacZ reporter-gene under control of the bovine and goat alpha-S1-casein genes with 5'-flanked sequences of various ex-tent: pCLZ1--721bp, pCLZ2-- 2001 bp and pCLZ3 3409 bp constructs. Five transgenic founders were generated by injection of the recombinant DNA into zygotes: pCLZ 1 - N 16, pCLZ2 - N 37 and pCLZ3 N 7, N 36, and N 48. Positive for J3-galactosidase activity cells were detected in lactating mammary glands of all transgenic females, however, distribution of the positive cells was variable. We observed two types of mosaics: clonal or "lobule" type with positive cells filling the whole of the globule or stochastic type with single positive cells scattered over one or different lobules. Two types of mosaics were characteristic of all the transgenic animals, although, females carrying the pCLZ2 transgene showed "lobule" type more often than transgenic animals with the transgenes pCLZ and pCLZ3. It is suggested that the stochastic type of mosaics occurs in the cells at terminal stage of differentiation, whereas the <> type arises from positive for P-galactosidase proliferating precursors. Analysis of the inheritance of the transgenes in different lines demonstrated that the pCLZl transgene was inserted in the X-chromosome of the founder whereas the other two localized in autosomes. Localization of the pCLZl transgene in the X-chromosome did not influence the mosaicism; it was similar to that of transgenic animals carrying the transgenes in autosomes. Ectopic expression of the reporter-gene was detected in mandibular glands from the offsprings of the founders N 16 and N 37 only, as well as in atrezed follicles in N 37. The weak ectopic expression saggests that the 5 S-flanked regulatory sequences used in the constructs are able to provide perfect tissue-specific expression of the reporter-gene.

[Secretion of biologically active human granulocyte colony-stimulating factor (G-CSF) in milk of transgenic mice].

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5' and 3' flanking promoter sequences of bovine alpha-S1-casein gene. Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3' flanking region of bovine gene @CSN1S1, but differed in size of the 5' flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 microg/ml to over 1 mg/ml, in mice with construct pGCml and was low (up to 60 microg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCml and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF.

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil.

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.

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.

[Effects of the nef and tat genes of the human immunodeficiency virus type I on rodent cells in vivo and in vitro]. / Vliianie genov nef i tat virusa immunodefitsita cheloveka tipa 1 na kletki gryzunov in vivo i in vitro.

The tat and nef regulatory genes of the human immunodeficiency virus type I (HIV-1) under the control of eukaryotic promoters were transferred in vivo into mice and in vitro into rat cell cultures. The development was disturbed and adenocarcinomas of the lacrimal glands and pancreas appeared in transgenic mice carrying the HIV-1 tat gene. Transfection with the tat gene altered morphology and increased proliferative activity of Rat-2 pseudonormal cells. The tat gene also induced the formation of neoplastic foci in a primary rat embryo fibroblast culture. The results obtained showed that the HIV-1 tat gene can act as an oncogene and activate the proliferation of cultured cells. Cell proportions in peripheral blood and bone marrow were altered and mitogen-induced lymphocyte proliferation was decreased in transgenic mice carrying the HIV-1 nef gene. This gene also significantly suppressed proliferation but had no effect on morphology of Rat-2 cells. Thus, the HIV-1 nef gene appeared to suppress proliferation of various animal cells.

[The level of sex steroids in the blood of inbred-strain mice during a normal pregnancy and after zygote retransplantation]. / Uroven' polovykh steroidov v krovi myshei inbrednykh linii vo vremia normal'noi beremennosti i posle retransplantatsii zigot.

The effect of genotypes on the blood content of progesterone and estradiol was studied in female CBA, C57BL and BALB mice. We also studied progesterone content at the early stages of pregnancy after the effect of stress factors, such as narcosis and surgery, involved in the retransplantation of the embryo from the donor to the recipient. The levels of sex hormones increased during pregnancy with two peaks on the 6-8th and 14-18th days. The pattern of changes in the level of both hormones depends on the female genotype: at some stages, reliable interstrain changes in the blood content of both progesterone and estradiol were found. At the same time, narcosis and bilateral laparotomy did not affect the blood level of progesterone within 12 h after mating, despite marked activation of the adrenocortical system. The data obtained should be taken into account when selecting mother-fetus pairs for retransplantation of embryos in experimental and farm animals.

[The effect of the micromanipulations used for transgenosis on mouse development]. / Vliianie mikromanipuliatsii, ispol'zuemykh dlia transgeneza, na razvitie myshei.

Non-specific effects of micromanipulation techniques used for producing transgenic mice on processes of embryonic development were studied. Zygotes obtained from C57BL and BALBxDD mice were treated as follows: (1) incubated in culture medium; (2) the male pronucleus punctured with a glass microneedle; (3) microinjected with a buffer solution; and (4) DNA (mouse P-35 oncogene with human insulin gene promoter) injected into the male pronucleus. Then zygotes were transferred into oviducts of syngeneic or allogeneic pseudopregnant females. Such treatment resulted in the intrauterine death of embryos, as well as in birth of the dead or non-viable offspring with numerous defects of development. Zygote pronucleus puncturing is the most damaging manipulation, since its effect exceeds that of the zygote incubation and is comparable with the effect of buffer of DNA injections.

[Different predisposition to the development of spontaneous and induced liver tumors in mice of different strains and their hybrids]. / Neodinakovaia predraspolozhennost' k razvitiiu spontannykh i indutsirovannykh opukholei pecheni u myshei raznykh linii i ikh gibridov.

No correlation between the sensitivity to induction of liver tumours by ortho-aminoazotoluene and predisposition to spontaneous development of these tumours was found in mice. Under crossing the predisposition to spontaneous tumours was inherited dominantly, while sensitivity to their induction was inherited by the intermediate type. Spontaneous hepatomas were more frequent in males, whereas induced onesin females of the same genotype.

[New data on the type of structural changes in DNA of simian adenovirus SA7 microinjected into mouse zygotes]. / Novye dannye o kharaktere strukturnykh izmenenii DNK adenovirusa obez'ian SA7, mikroin''etsirovannoi v zigoty myshei.

The structure of the transgene has been analysed in a new series of experiments on the transfer of adenovirus SA7 DNA into the mice zygotes by microinjection technique. The previous data on SA7 DNA elimination from the genomes of different organs (sceletal muscles, heart, tail) have been confirmed and detailed for the F0 and F1 generations of transgenic animals. The left end of adenoviral genome has been shown to be predominantly transfered after microinjections of SA7 DNA into the mice zygotes.

[Production of transgenic rabbits and mice carrying the gene for bovine growth hormone]. / Poluchenie transgennykh krolikov i myshei, soderzhashchikh gen gormona rosta byka.

The results of experiments on the transfer of bovine gene for growth hormone into mice and rabbits are presented. The gene was transferred by the technique of microinjection into the zygote. In all cases transgene in rabbits occurred to be changed. In two transgenic mice the bovine growth hormone gene represented some tandem arranged copies. One of the mice had accelerated growth. This phenotypic changes is found to be inheritable.

[Distribution of alpha 2M-globulin and Lpm-protein in the organs and tissues of the American mink]. / Raspredelenie al'fa 2M-globulina i Lpm-proteina v organakh i tkaniakh amerikanskoi norki.

Distribution in the organs and tissues of two proteins of alpha-macroglobulin fraction, that differ in their antigenic structures, has been studied in the American mink. The both proteins (alpha 2M and Lpm) are present in hepatocytes, in cells of the follicular epithelium of the ovary, in the thymic bodies, in the alveolar macrophages of the lungs and in the splenic lymphoid nodes. Joint localization of alpha 2M and Lpm is revealed in the connective tissue of all the organs examined. The exception make the stomach and the uterine, where alpha 2M is revealed but not Lpm. The results obtained demonstrate a similar distribution of the two alpha-macroglobulins in the mink organism. They correspond to the literature data on morphofunctional topography of alpha 2M in the man. Certain individual differences in alpha 2M and Lpm localization can reflect peculiarities inherent in each of these alpha-macroglobulins of the American mink.