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.

 

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