The basis microinjection buffer to obtain transgenic animals
(57) Abstract:The invention relates to genetic engineering, in particular the production of transgenic animals. The application of the synthetic environment of the fallopian tubes MSOF as the basis microinjection buffer to obtain transgenic animals. Microinjection solution based MSOF is more physiological, it has less impact on the viability of embryos. 3 table. The invention relates to genetic engineering, in particular the production of transgenic animals.Known standard buffer TE (10 mm Tris-HCl, pH of 7.4, 0.1 mm EDTA, pH 8.0), which is used for cooking microinjection solutions gene constructs with the aim of obtaining transgenic animals (Hammer, R. E., Pursel, V. G., Rexroad C. E. Jr. that is, a. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985, 315, 680-683). Buffer THOSE characterized by a very low osmotic pressure (~25 mOsm) in comparison with the majority of media for embryo culture, which is one of the reasons for the relatively low viability of injected embryos.Known synthetic environment of the fallopian tubes to buffer the basis pug (MSOF) for microinjection of embryos of cattle and study of their vegatative. The injection of a physiological buffer MSOF did not affect the development of blastocysts in vitro. The proportion of embryos that developed to the blastocyst stage when injected MSOF, was two times higher in comparison with injection buffer (respectively 27.5% and 13.9%) and did not differ from control penjelasannya embryos (27,6%). The ability to use the environment MSOF to obtain transgenic animals has not been established.The aim of the present invention is the use of synthetic environment of the fallopian tubes MSOF as the basis microinjection buffer to obtain transgenic animals.To prepare microinjection solutions MSOF and THOSE used filtered (0.22 μm) bidistilled water. Buffer TE (10 mm Tris-HCl, pH to 7.4; 0.1 mm EDTA, pH 8.0) was prepared and kept in the form of a 2-fold solution (2x). In the preparation of injection solution gene constructs concentration of the buffer was brought to 1x.The composition of MSOF indicating polyarnosti initial solutions and their final concentrations in 1x buffer is shown in table 1. Concentrated solutions of the individual components of this buffer was prepared in advance and kept at +4oC or at -20oC (L-glutamine, streptomycin, penicillin) for three months. The day before, e is actor gene constructs and drove concentration MSOF to 1x. The concentration of DNA in injection solutions MSOF and they were set at 1000 copies/PC. Microinjection the solutions were stored at +4oC and used within one day.Example. The experiment was carried out on rabbits. These animals are well developed methods of superovulation, extraction, microinjection and transplantation of embryos. Rabbits have a short gestation period, so the results can be obtained within a short period of time (months). The patterns revealed in rabbits can be transferred to other species of farm animals.Zygote rabbits were obtained from superovulated females rabbits breed Zika. Preparation of donors and recipients, retrieval and transplantation of embryos was performed in a known manner (Zinoviev N. A., Besenfelder U., Brehm, Obtaining eggs and transplantation microinjection zygotes in rabbits. Ontogeny, 1996, 27(3): 214-217). Microinjection was carried out in male pronucleus under a microscope at magnification h. In each embryo were injected with 1-2 PCL DNA solution. Until transplantation microinjection embryos were cultured in a drop of culture medium (PBS+20% serum) at +37oC for 1-2 hours. As a control part recipientprivatekey diagnosis of pregnancy. Kindling is expected on the 31st day after the transplantation of embryos. In case of delay recipients were injected with 0.3 IU of oxytocin intramuscularly. Tissue samples (ear) was collected on day 4-5 after kindling, while labeling rabbits with a marker. This marking was maintained for 1-2 weeks, which was enough for analysis and diagnosis of transgenetic. Identified transgenic animals again were marked with a marker at the age of 2-3 weeks and then finally (ear marker) at the age of 1-1,5 months.DNA from tissue samples was isolated using a modified method of phenol-CHLOROFORMATES extraction (Ausubel F. M., Brent R., Kingston R. E. e. a. Current protocols in molecular biology. New York, 1987). Diagnosis of transgenetic performed by capillary polymerase chain reaction (PCR) (Zinoviev N. A., Ernst, L. K., Brehm, Method capillary polymerase chain reaction as a rapid and cheaper alternative proof of different DNA variants. In Proc.: Genetic engineering of agricultural animals. M., 1995: 244-248). PCR products were separated by 1.6% agarose gel in TBE buffer (0.1 M Tris-HCl, pH 7.5; 0.1 M boric acid; 2 mm EDTA, pH 8.0) with the addition of the bromide dimidia to a final concentration of 30 ng/µl, viewed under ultraviolet light and fotografierte presented in table 2. From the obtained data (table. 2) it follows that the use of buffer MSOF increased the share of normal kindling by 12.8% compared with THE buffer (p < 0,95). On average each okruglivshimsya recipient in the group MSOF was obtained at 1.2 Bunny compared with THE group (p > 0,99), while the number of embryos, the average transplant recipients in the experimental groups did not differ (of 20.1 and 20.3). Thus, MSOF was less harmful to embryos compared to THOSE. In the group MSOF was only 20% of the recipients, which brought in 1-2 Bunny, while in THE group was 50%. The proportion of stillbirths in the group MSOF was 16.2% lower compared with THE group (p > 0,999). The main physiological indicator, which directly allows to determine the influence of injection buffer on the viability of embryos, is the survival rate of embryos. In the group MSOF noted in 1.7 times higher overall survival rate of embryos in comparison with THE group (p > 0,999). The calculation of this indicator for okruglivshimsya recipients showed that the survival rate of embryos injected MSOF, was higher compared with THOSE in 1.4 times (p > 0,999). Therefore, the injection buffer on the basis of MSOF differs from the standard injection buffer THE best physiological prospect who's table it, between research groups there were no differences in the degree of integration (9.0 and 9.8 per cent), because integration depends primarily on the method used for introducing DNA into embryonic lines mammals, and species. Physiological parameters with the degree of integration of the transgene characterizes the measure of the overall efficiency. It is known that in rabbits it is approximately 1%. When transferring the same number of embryos due to their better survival is born a larger number of animals, among which, with an equal degree of integration there is a larger number of transgenic animals. In these studies due to the use of more physiologically suitable buffer MSOF the overall efficiency of gene transfer increased 1.6 times (p < 0.95) and was 1.4% compared to 0.9% when using THOSE.The indicator of efficiency of gene transfer in okruglivshimsya recipients eliminates the influence of those recipients who, at the time of the experiments were not able to implantation of the embryos and their subsequent development. The calculation of this index revealed differences in 1.3 times (p < 0,95) in favor of MSOF. In addition, the group MSOF received more transgenic animals, only 14.3% of them b the ü 2 times more individuals (28.6 per cent).Based on these results, we can conclude that the environment MSOF as the basis microinjection buffer is more suitable for use in obtaining transgenic rabbits compared with standard buffer. MSOF has less of an impact on the viability of embryos and as a result contributes to a higher efficiency of transgenesis in rabbits. It is known that similar patterns of change in physiological parameters detected during cultivation in vitro of bovine embryos. In this regard, we can suggest the best suitable solution MSOF to obtain transgenic cattle and other animal species.The invention is applicable in biotechnology. The synthetic environment of the fallopian tubes MSOF as the basis microinjection buffer to obtain transgenic animals.
SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.
EFFECT: enhanced effectiveness in producing living descendants.
39 cl, 5 dwg, 1 tbl
SUBSTANCE: the present innovation deals with individual matching donor sheep to recipient sheep at similar antigenic composition of blood types being correspondent to the value of antigenic similarity index being ra=0.51-1.00, where ra - antigenic similarity index. Moreover, the mentioned antigenic similarity index should be calculated by the following formula: where S - the number of similar antigens in a donor sheep and in a recipient sheep, n1 - the number of detected antigens in a donor sheep, n2 - the number of detected antigens in a recipient sheep. The present innovation enables to increase the level of adaptability of transferred ovine embryos by 25%.
EFFECT: higher efficiency of embryo transfer.
3 ex, 4 tbl
SUBSTANCE: method includes ovary removal after animal slaughtering, their transportation, ovocytes extraction from ovary, their cultivation to metaphase II stage, preparation of boar sperm, combined cultivation of ovocytes and spermatozoids and cultivation of ovulums. Besides, ovary is separated from animals no later than 30 minutes after the moment of animal immobilisation. Ovary is transplanted in free of antibiotics salt solution at 35-37°C during 1 hour. Ovocytes are taken from ovary by dissectioning of visible antrum-containing follicles. Received ovocytes are split into groups by quantity of surrounding cumuluce cell layers, namely: ovocytes surrounded by 4-5 layers of cumuluce cells, 2-3 layers and one layer of cumuluce layer. Each group of ovocytes is cultivated in medium containing 10 IU of human chorionic gonadotropin. To fertilise ovocytes in vitro, ejaculated sperm of pigs is used, which is dissolved with glucose-chelate-citrate-sulphate diluter and cleaned from semen plasma dissolved with mTBM (mouse thymic virus) medium and incubated during 25 minutes under incubator conditions at 5% CO2 and 38.5°C. Produced zygotes are cultivated in NCSU-23 medium free of phenol red and enriched with 0.1% of amino acids during 6-7 days; in addition, 0.17 mM Na-pyruvate and 2.75 mM Na-lactate are introduced into NCSU-23 medium during the first three days of cultivation.
EFFECT: increase of mature ovocytes yield and their fertilisation ratio in vitro.
1 tbl, 1 ex
FIELD: package industry.
SUBSTANCE: container 10 includes vessel (21) with biological medium, germinal cells and/or one or more embryos. Vessel (21) has CO2 permeable wall, CO2 permeable gasket (28) and closure medium (30) for selective access to internal vessel cavity. Buffer chamber (60) for air saturated with CO2 surrounds vessel at least partially and is formed by shell (61). Buffer chamber is connected to CO2 permeable wall. Such container construction is intended specifically for intravaginal cultivation, and permeable gasket (28) prevents vaginal secretion from entering buffer chamber. Buffer chamber (60) normalises water pH in vessel after the vessel leaves environment saturated with CO2.
EFFECT: prevented negative effect for long-term embryo(s) cultivation.
53 cl, 19 dwg
SUBSTANCE: method includes oophorectomy in animals after slaughter, transportation of oophorons, extraction of oocyte-cumulus complexes by aspiration of follicles content, irrigation of the complexes and their cultivation in a medium not covered with oil, in the presence of serum gonadotropin of pregnant mares, human chorionic gonadotropin and 5% estrous serum of bovine cattle. At that, follicles of diametre from 2 to 8 mm are used to obtain oocytes. After extraction of the oocyte-cumulus complexes are immediately placed in a washing medium containing 0.5 mM of isobutylmethylxanthine to synchronise reinitiation of meiosis. Before the cultivation the selection of oocytes based on their morphological evaluation is carried out. All the procedures of isolation, irrigation and selection are carried out in a medium containing 0.5 mM of isobutylmethylxanthine. Oocyte-cumulus complexes were cultured in a medium modified by insertion of 50 ng/ml of bovine prolactin hormone.
EFFECT: increase of proportion of mature oocytes in vitro, and their ability for further development.
1 ex, 1 tbl
SUBSTANCE: method comprises extracting the oocytes-cumulus complexes from ovaries, cultivating them to the stage of metaphase II, co-cultivating of oocytes and sperm cells, cultivating the impregnated ootids. And additionally the follicle-stimulating and luteinising hormone is administered in the form of the combined preparation Menopur, estradiol, heparin, caffeine and antibiotic. The cultivating is carried out in the base medium. Extraction of the oocytes-cumulus complexes from ovaries is carried out from live sheep under anaesthesia by puncturing ovarian follicles during laparotomy. Superovulation stimulation is preliminary carried out. In the maturation medium the preparation Menopur is added, the concentration of both components that are part of the preparation, is 0.075 IU/ml, and estradiol is added to the maturation medium in the concentration of 10 mcg/ml. For impregnation the newly obtained sperm of tups is used. For tup sperm capacitation and to the maturation medium the combination of heparin is added in an amount of 5 units/ml and caffeine in an amount of 0.2 mg/ml. The cultivation and impregnation is carried out in four well Petri dishes in 500 mcl nutrient medium under 200 mcl paraffin oil.
EFFECT: use of the method enables to increase the proportion of output of oocytes.
3 cl, 11 dwg, 3 ex