AGRIS - 

2023

Donnik, I.M. | Makutina, V.A. | Krivonogova, A.S. | Isaeva, A.G. | Dejkin, A.V. | Koshchaev, A.G.

The results of research into genetic methods of breeding agricultural animals with improved characteristics are presented. A significant number of animals with an edited genome have been obtained. Methods of genome modification in cattle are constantly improving. The approaches of gene editing of bovine embryos, delivery of edited constructs and improvement of embryo survival after introduction of editing systems have been studied. The studies were performed on cattle embryos. BLG and SD209 gene editing systems were developed and approbated. Delivery variants of the editing system into cattle cells were studied: microinjection into the zygote of plasmid DNA encoded sequence with CRISPR/Cas9 c sgRNA, the method of viral vectors (adeno-associated AAV viruses, serotypes AAV1, AAV2, AAV6, AAV9, AAVDJ), co-injection of plasmid DNA and sperm into the oocyte at the MII stage, and microinjection of Cas9 and guide RNAs. Different techniques for performing micro-injections have been investigated and refined, and different variants of preparation of Cas9 RNA and guide RNA mixtures have been tested. Based on these results, the protocol for performing microinjection of the editing system was optimized and a model experiment was performed on 160 oocytes, with 80 cells per each construct. The findings have shown that the efficiency of editing has generally increased. When injected with guide RNA against BLG gene and spCas9 mRNA, 84% of the surviving cells initiated cleavage, blastulation was 20%, and BLG knockout was 69.2%. When injected against the CD209 gene and spCas9 mRNA, 44.4% of the surviving embryos started cleaving, blastulation was 16.7%, with CD209 knockout at 44.4%. The novelty of the work lies in obtaining data on the development of editing systems with specific target genes, in improving the delivery system and cultivation of bovine embryos. | Представлены результаты исследования генетических методов создания с.-х. животных с улучшенными характеристиками. Получено значительное количество животных с отредактированным геномом. Методы модификации генома у крупного рогатого скота (КРС) постоянно совершенствуются. Изучены подходы генного редактирования эмбрионов крупного рогатого скота, доставки отредактированных конструкций, повышения выживаемости эмбрионов после внесения систем редактирования. Исследования проводили на эмбрионах КРС. Разработаны и апробированы системы редактирования генов BLG и СД209. Изучены варианты доставки системы редактирования в клетки КРС: микроинъекция в зиготу плазмидной ДНК с закодированной последовательностью с CRISPR/Cas9 c sgRNA, способ вирусных векторов (аденоассоциированные вирусы AAV, серотипы AAV1, AAV2, AAV6, AAV9, AAVDJ), совместное введение плазмидной ДНК и сперматозоида в ооцит на стадии MII, а также микроинъекции РНК Cas9 и гидовых РНК. Исследованы и усовершенствованы различные методики выполнения микроинъекций и опробованы различные варианты приготовления смеси РНК Cas9 и гидовых РНК. На основе полученных результатов оптимизирован протокол выполнения микроинъекции системы редактирования и проведен модельный эксперимент на 160 ооцитах, по 80 клеток на каждую конструкцию. В результате установили, что эффективность редактирования в целом повысилась. При инъекции гидовой РНК против гена BLG и мРНК spCas9 дробление начали 84% выживших клеток, бластуляция составила 20%, оказались с нокаутом по BLG – 69,2%. При инъекции против гена CD209 и мРНК spCas9 дробление начали 44,4% выживших эмбрионов, бластуляция составила 16,7%, с нокаутом по CD209 – 44,4%. Новизна работы заключается в получении данных о разработке систем редактирования с определенными генами-мишенями, в усовершенствовании системы доставки и культивирования эмбрионов КРС.

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Central Scientific Agricultural Library - Russian Federation

2021

Narovlyanskij, A.N. | Sanin, A.V., The N.F. Gamalej Research and Development Inst. of Epidemiology and Microbiology, Moscow

A pharmacokinetic study of the absorption into the bloodstream, bioavailability and excretion of Gamavit from the body after intramuscular administration to 6 laboratory mini-pigs was conducted. Quantitative determination was carried out by high performance liquid chromatography (HPLC) using a fluorimetric detector, for which Gamavit was labeled with Су5 dye, which was then used for mini-pigs inoculation. Gamavit contains a complex of active substances in the form of sodium nucleinate in an amount of 0.02 mg/ml and acidic hydrolysate of denaturated emulsified placenta in an amount of 0.5 mg/ml. The developed methods for determining Gamavit in the blood and feces were validated according to the following validation parameters: selectivity, calibration curve, accuracy, precision, limit of quantitative determination, sample transfer, and sample stability. The confirmed analytical range of the method for Gamavit detection in blood plasma and feces was 1.00-50.0 mcg/ml. Maximum concentration of Gamavit in the blood of mini-pigs after a single intramuscular injection was 30.97 mcg/ml and was reached on average 15 min after administration. 24 h following administration, Gamavit was still detected in the blood in insignificant amounts. The average half-life of Gamavit in the blood is 8.64±3.50 h. After administration at a dose of 0.1 ml/kg, the clearance of the drug is 1.27 l/kg*h, the excretion rate at an effective concentration of 30 mg/l is 38 mg/kg*h, and the maintenance dose when using the drug 1 time a day is 0.9-1.0 ml. The detection of the label in the feces of the studied animals indicates that one of the ways Gamavit removal is excretion with bile acids, as well as partial excretion with feces. | Проведено фармакокинетическое исследование всасывания в кровоток, биодоступности и выведения из организма Гамавита после внутримышечного введения 6 лабораторным мини-свиньям. Количественное определение проводили методом высокоэффективной жидкостной хроматографии (ВЭЖХ) с использованием флуориметрического детектора, для чего Гамавит метили красителем Су5, который потом и использовали для введения животным. Гамавит содержит комплекс действующих веществ в виде нуклеината натрия 0,02 мг/мл и кислотного гидролизата плаценты денатурированной эмульгированной 0,5 мг/мл.Разработанные методики определения Гамавита в крови и кале животных были валидированы по следующим валидационным параметрам: селективность, калибровочная кривая, точность, прецизионность, предел количественного определения, перенос пробы, стабильность образцов. Подтвержденный аналитический диапазон методики составил 1,00…50,0 мкг/мл для Гамавита в плазме крови и кале. Установлено, что максимальная концентрация Гамавита в крови животных после однократного внутримышечного введения составляет 30,97 мкг/мл и достигается в среднем через 15 мин после введения. Через 24 ч после введения Гамавит все еще определяется в крови в незначительных количествах. Средний период полувыведения Гамавита по крови составляет 8,64±3,50 ч. При введении Гамавита в дозе 0,1 мл/кг клиренс препарата равен 1,27 л/кг*ч, скорость выведения при эффективной концентрации 30 мг/л равна 38 мг/кг*ч, а поддерживающая доза при применении препарата 1 раз в сутки составляет 0,9…1,0 мл.Обнаружение метки в фекалиях исследуемых животных свидетельствует о том, что одним из способов выведения препарата Гамавит является выведение с помощью желчных кислот и частично он выводится с калом.

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Central Scientific Agricultural Library - Russian Federation

2018

Afonyushkin, V.N., Siberia Federal Scientific Center of Agrobiotechnology, Novosibirsk Region (Russian Federation) | Shirshova, A.N. | Shamovskaya, D.V. | Plomod'yalov, D.N. | Kozlova, Yu.N., Russian Academy of Sciences. Siberian Branch, Novosibirsk (Russian Federation). Institute of Chemical Biology and Fundamental Medicine

In spite of applying specific prophylaxis the occurrence of avian infectious bronchitis (AIB) caused by Coronavirus is progressively increasing. It is difficult to use antiviral drugs for treatment this disease in commercial poultry breeding. The effectiveness of suppressing reproduction of the AIB virus in bronchi and trachea of 2-week broiler chickens with Triviron based on synthetic ribonuclease was studied. The broilers were treated with an aerosol of 1% solution of this drug 3 times with an interval of 1 day with exposure of 10, 20, 40 or 60 min in a dose of 3ml/m3. The control group included 7 chickens; each of 4 test groups included 6 chickens. A statistically significant decrease in the concentration of the agent after the treatment of the chicks was established within 40 min. To estimate the aerosol accessibility with a drop diameter of about 10 um, the dynamics of accumulation of aerosol particles of marker DNA molecules in the trachea and bronchi was studied. The yield of the growth curve of the concentration of DNA marker on the plateau in the trachea began after 20 min exposure of the aerosol with. After a 40 min exposure the concentration of marker DNA in the bronchi increased. The coincidence of the dynamics of accumulation of aerosol particles in the bronchi and trachea with data on the reduction in the concentration of the IBC virus at different exposure confirms the consistency of the developed model for evaluating the effect of Trivirone on the AIB virus in the body of broiler chickens. The preparation and the method of its application are recommended for the treatment and prevention of AIV in broiler chickens. | Несмотря на широкое применение специфической профилактики, частота встречаемости инфекционного бронхита кур (ИБК), вызываемого вирусом р. Coronavirus, увеличивается. Применять противовирусные средства при данном заболевании в условиях промышленного птицеводства затруднительно. Изучена эффективность подавления репродукции вируса ИБК в бронхах и трахее 2-недельных цыплят-бройлеров препаратом Тривирон на основе синтетической рибонуклеазы. Птицу обрабатывали аэрозолем 1%-ного раствора препарата аэрозольно 3 раза с интервалом в 1 сутки с экспозицией 10, 20, 40 или 60 мин. в дозе 3 мл/м3. В контрольную группу было включено 7 цыплят, в 4 опытные – по 6. Установили статистически достоверное снижение концентрации агента после обработки цыплят в течение 40 мин. Для оценки доступности аэрозоля с диаметром капель около 10 мкм изучили динамику накопления маркерных молекул ДНК, которые вводили аэрозольно, при разных экспозициях, в трахее и бронхах. Выход кривой роста концентрации маркерных молекул ДНК на плато в трахее начинался с 20-й минуты экспозиции аэрозоля. После 40-минутной экспозиции концентрация маркерных молекул ДНК в бронхах повысилась. Совпадение динамики накопления частиц аэрозоля в бронхах и трахее с данными о снижении концентрации вируса ИБК при разных сроках экспозиции подтверждает непротиворечивость разработанной модели оценки действия Тривирона на вирус ИБК в организме цыплят-бройлеров. Препарат и способ его применения рекомендованы для лечения и профилактики ИБК у цыплят-бройлеров.

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Central Scientific Agricultural Library - Russian Federation

2012

Chernikova, D.A. | Rogozin, I.I. | Managadze, D., National Institutes of Health, Bethesda

From position of modern genomics the complexity of the eukaryote genome organization, including structural genes, genes of transport and ribosomal RNA, the tandem and intersperced repeats is discussed in details. The scientific data testifying high frequency of occurrence in genome sequences of farm animals (cattle, horse) the sequences of different classes of endogenous retroviruses, and also high frequency of transpositions of mobile genetic elements (MGE) in comparison with mononucleotide replacements are provided. The paradox between rather small quantity of nucleotides in the genomes falling on structural genes, coding proteins (about 2% from a haploid genome) in mammalian genomes, and the big size of transcriptome including transcripts more than 90% genome is considered. The concept of "a dark matter" and participation in its formation of long non-coding RNA was considered. The regulatory role in a gene expression of long non-coding RNA and their communication with MGE was analyzed. On modeling objects (genomes of a mouse, human) the comparative analysis of the frequencies of Long Interspersed Nuclear Elements (LINE) localizations, which transcribed in long non-coding RNA, in gene regions being under different pressure of natural selection has been carried out. Convincing data are obtained, testifying that the frequency of LINE localization is statistically authentic below in exons in comparison of promotors, and below in promotors in comparison of introns, that corresponds to known intensity differences in control of these sequences by factors of natural selection. Considering the high speed of transpositions (10-2 transpositions on one transposon, on one generation) the complexity of evaluation of the genetic and adaptive contribution within N.I.Vavilov's law on homological ranks of hereditary variability in relation with the MGE accumulations in genomes of highest plants and animals is analyzed. Existence of "clearing" selection in relation to "ancient" MGE (LINE) in long non-coding RNA is discovered | С современных позиций анализируется сложность организации генома эукариот, включающего структурные гены, гены транспортных и рибосомальных РНК, тандемные и диспергированные повторы. Приводятся литературные данные, свидетельствующие о высокой частоте встречаемости в секвенированных последовательностях геномов с.-х. видов (крупный рогатый скот, домашняя лошадь) последовательностей разных классов эндогенных ретровирусов, а также высокая частота транспозиций мобильных генетических элементов (МГЭ) по сравнению с мононуклеотидными заменами. Обсуждается парадоксальность отношений малого количества нуклеотидов в геномах, приходящихся на структурные гены, кодирующие белки (около 2% от гаплоидного генома) в геномах млекопитающих, с величиной транскриптома, в который входят транскрипты более 90% от всего генома. Рассматриваются понятие "темной материи", участие в ее формировании длинных некодирующих РНК, а также регуляторная роль в генной экспрессии длинных некодирующих РНК и их связь с МГЭ. На модельных объектах (геномы мыши, человека) выполнен сравнительный анализ частот локализаций транскрибируемых в длинные некодирующие РНК последовательностей длинных диспергированных ядерных элементов (LINE) в участках структурных генов, находящихся под разным давлением естественного отбора. Частота локализации LINE статистически достоверно низкая в экзонах, более высокая в промоторах, и наибольшая — в интронах, что соответствует отличиям в интенсивности контроля этих последовательностей факторами естественного отбора. Учитывая высокую скорость транспозиций (10-2 транспозиций на один транспозон на одно поколение) рассматриваются сложности оценок вклада генетической и адаптивной компонент в гомологические ряды наследственной изменчивости Н.И. Вавилова в связи с накоплением данных о доминирующей роли МГЭ в организации геномов высших растений и животных. Отмечается наличие "очищающей" селекции по отношению к "древним" МГЭ (LINE) в длинных некодирующих РНК.

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Central Scientific Agricultural Library - Russian Federation

2011

Arkhipov, A.V. | Vishnichenko, V.K., All-Russia Research and Development Inst. of Agricultural Biotechnology, Moscow

For the first time in the world literature the authors presented the evidence of expression in ballot plants (Allium сера L. var. aggregatum G. Don) of nucleotide sequences coding the polypeptides omologous to known plant Dicer-like protein (DCL), obtained by PCR, sequencing and computer lalysis. The investigated polypeptides contain specific for DCL-protein the dsRNA bind, PAZ and IBOc domens. The solution of assigned task is available only with use of diagnostic oligonucleotide rimers, the design of which was accomplished on the basis of information on EST-sequences (expressed sequence tags) in Allium sativum (GarlicESTdb integrated database). The results permit to taw a conclusion about high extent of divergence of DCL-proteins in Arabidopsis, poplar, rice, on he one hand, and the plants of Allium genus, on the other hand, that is revealed on level both nucleotide sequences of mRNA, and amino acid sequences of studied polypeptides | Впервые в мировой литературе представлены полученные с помощью методов полимеразной цепной реакции, секвенирования и компьютерного анализа доказательства экспрессии в растениях лука-шалота (Allium сера L. var. aggregatum G. Don) нуклеотидных последовательностей, кодирующих полипептиды, гомологичные известным растительным DCL-белкам (Dicer-like). В составе исследуемых полипептидных цепей выявлены характерные для молекулы DCL-белка домены dsRNA bind, PAZ и RIBOc. Решение поставленной задачи оказалось возможным только при применении диагностических праймеров, дизайн которых базировался на использовании информации по EST-последовательностям (еxpressed sequence tags) у Allium sativum (база данных GarlicESTdb). Результаты позволяют сделать вывод о высокой степени дивергенции DCL-белков арабидопсиса, тополя и риса, с одной стороны, и растений рода Allium — с другой, что выявляется на уровне как нуклеотидных последовательностей мРНК, так и аминокислотных последовательностей исследуемых полипептидов

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Central Scientific Agricultural Library - Russian Federation

2009

Timerbaev, V.R. | Dolgov, S.V., All-Russia Research and Development Inst. of Agricultural Biotechnology, Moscow

In order to increase the shelf-life of biotechnological tcmato fruits, binary vectors that trigger the process of RNA interferention have been constructed. A tomato gene for ACC oxidase, a key enzyme of ethylene biosynthesis, was selected as target. The gene construction was based on a full-size sequence of the 4 exon of the 496-bp gene LeACO1. The effect of orientation of the repeats on the efficiency of RNA iterferention was investigated. To this end, two possible expression cassettes with opposite relative positions of the repeats, antisense-sense and sense-antisense were designed. As a result of agrobacterial transformation of the line Yalf tomato, 28 and 19 lines with each of the above orientation, respectively, were obtained. The insertion of a selective gene for kanamycin resistance was confirmed in all lines by PCR, whereas the occurrence of the target insertion was determined in 27 and 18 lines, respectively. A significant delay in fruit ripening in the majority of the analyzed lines with antisense-sense DNA-fragment orientation was observed; the fruits harvested at the blange stage retained good quality during 3-4 months of storage. The fruits with the opposite orientation had the same maturation characteristics as in the control plants. We also observed the dynamics of the development of fruit color during storage under controlled conditions for 140 days.

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Central Scientific Agricultural Library - Russian Federation

2007

Fevet, G. | Tencheva, D.

The infectivity of bee queens and drones with deformed wing virus (DWV), as well as its distribution within different organs and tissues were studied by the RT-PCR method, immunochemistry and electronic microscopy. DWV was detected in 67% of clinically healthy productive bee queens, 78% of drones collected at a hive entrance and 100% of drones before the hive entrance. The number of virus genome copies in RT-PCR was maximum in testes and digestive tracts of drones, lower – in glands; in queens – maximum in ovaries and the least – in heads and digestive tracts. Some attempts to identify the localization of DWV in digestive tracts and ovaries by a serological test and hybridization were not successive in spite of detection of virus RNA in RT-PCR. Adipose cells gave a strong and specific response to DWV. Therefore, the DWV infection of fatty cells in queens results in their reduced productivity. In drones DWV was detected in epithelial cells in seminal vesicles, accessory glands. The intensive replication of DWV in them influenced negatively the productivity of drones, and consequently contaminated sperm was a source of infection bee females and the next bee generations. DWV was also detected in most drones’ epithelial cells of proventriculus, mid and hind intestine. It is concluded that DWV affects certain tissues of bees’ genital and digestive tracts. Many epithelial cells are adjacent to the basal membrane which is a physiological barrier preventing from penetrating viruses into internal organs. This explains the differences in the infection running under peroral and vector infections of bees. The spread of infection in a bee family among adult bees and larvae in the nutrition process or through gland excretions is much lower.

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Central Scientific Agricultural Library - Russian Federation

1995

Tyupaev, I.M.

Problems of formation and regulation of meat animal productivity with reference to age dynamics of three key protein metabolosm processes in sceletion muscles -synthesis, degradation and accretion, are discussed. The number of protein synthesized in selection muscles appeared to be determined by fund of total RNA and its activity. This activity of total RNA in selection muscles is rather steady in case of permanently adequate animal rationswith respect to their requirements for upkeep during breeding. Vital role in ratio regulation for chemical components of tissue growth in the same breed animals is attributed to the number of nutrient absorbed relative to requirements for upkeep of metabolism and protein accretion. Regulation of the growth efficiency may be resulted from prolonged animal development period in case of peak stimulus to growth. Physiological growth regulation is more promising to increase bioenergetic growth efficiency compared to alterations of animal genotypes

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Central Scientific Agricultural Library - Russian Federation

1994

Borisenko, A.S. | Shayakhmetov, D.M. | Gerzilova, A.G. | Tikhonenko, T.I.

The possibility was studied to use the U3 promoter from LTR region of bovine leukemia virus to control the expression of the antisense RNA genes directed against the BLV genome in cell cultures FLK-BLV, CC81, and HeLa. The genetic engineering constructions were obtained carrying the reporter beta-galactosidase gene and the genome of antisense RNA to R-U5 region of the viral genome under the control of the promoter. The functioning of the viral promoter was studied in three cell lines. Its specific activation and kinetics of inhibition of BLV virus reproduction in cell culture CC81 have been demonstrated. The maximal 75% level of the viral reproduction inhibition was achieved at threefold molar excess of the plasmid containing the antisense RNA gene over the pregenomic viral DNA

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Central Scientific Agricultural Library - Russian Federation

1993

Balashvili, M.I. | Todadze, M.I. | Kvesitadze, G.I. | Dzhokhadze, D.I.

The effect of the preparation Aminol-forte manufactured by Inagrosa (Spain) on the endogenous transcriptional activity of cell nuclei and chloroplasts in leaves of some legumes (horse bean, kidney bean and soya bean) was studied. It is shown that a two- or three-fold sprinkling of leaves during the first ten days after the appearence of sprouts significantly stimulated the primary manifestation of genetical activity of the organelles, i.e. their ability to synthesize RNA. Both nucleolar and carioplasmic transcription increased in nuclei after the treatment. It is suggested that the stimulating effect may result from the enhancement of the activity or amount of DNA-dependent RNA-polymerase, of from genome deblocking

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Central Scientific Agricultural Library - Russian Federation