Method for in vivo transformation of animal testicle stem cells

FIELD: biotechnology.

SUBSTANCE: claimed method includes injection of vector construct pX-RSVhGH encoding human growth hormone or pX-Ins, encoding human insulin into parenchyma of 8-9-week rabbit males. Transformation effectiveness of gene constructs is determined by immunohystochemical kit Novocastra (Sigma, USA).

EFFECT: method for genetic transformation of rabbit testicle stem cells with increased effectiveness.

3 dwg, 1 ex

 

The invention relates to biotechnology, in particular genetic engineering, and in particular to methods of transformation of stem cells of the testes (SCS) rabbits in vivo by introduction of a foreign genetic material in the form of vector systems.

There are many methods of genetic transformation of cells of the testes. Most of them belong to the transformation of cells in culture, or to obtain transgenic animals. So, for example, as a promising technique introduction cdnc in sex cells using electroporation method, based on the influence of an external electric field, contributing to the formation in the cell membrane transient pores through which exogenous DNA enters the cell (Baranov V.S., Gopala I., Grech P. etc. the Inclusion of macromolecules in the male germ cells of mice using electroporation and dimethyl sulfoxide // Cytology and genetics. - 1990. - 24. No. 3. - P.3-7; Kuznetsov A.V., Veerapen. The penetration of exogenous DNA into competent spermatozoa Mytilus Galloprovincialis Lam. // The ecology of the sea. - 2001. - W. - P.76-79; Z. Yu, R. Guo, Y. Ge et al. Gene expression profiles in different stages of mouse spermatogenic cells during spermatogenesis // Biol. Reprod. - 2003. -V.69(1). - P.37-47). This method allows the use of a common physical nature of passage of electrical impulses through all biological membranes and allows you to enter cdnc in cells of any species and TC is Navaho origin. For transfection cells often use plasmid pCMVlacZ, containing the bacterial lacZ gene of E. coli. The choice of this vector is determined by the ease of detecting its expression in the cells. With the introduction of cdnc using electroporation in the Mature added mouse revealed that immature forms of the spermatogenic epithelium able to incorporate labeled cdnc under the influence of electric shock. During short-term exposure to electric current with options 400 V and 500 μf efficiency transfection of SCS is low and amounts to only 1.3%. The disadvantages of the method of electroporation should also include the need for special equipment.

There is a method of genetic transformation by introducing the genetic material in the testes of animals in vitro by liposomal transport genes (Bachiller d, Schellander K., Peli J. et al. Liposome-mediated DNA uptake by sperm cells // Mol. Reprod. Dev. - 1991. -V.30(3). - P. 194-200). As a vector for the transfer of exogenous DNA extracted stem cells from the testes of animals and manipulation in vitro (incubation with liposomes containing plasmid DNA). After obtaining transgenic sperm artificially osumenyi females. It was established that the transgene was transmitted to the F1 generation of rabbits. Among the shortcomings of liposomal transport genes should be noted cytotoxicity of liposomes, as well as Otsu is due, in most cases, integration of transgenes (up to 0.8%).

As the prototype was taken method mediated by retroviral transfection of germ cells of the testes of adult boars (Nagano M, Shinohara T., Avarbock M.R., and Brinster R.L. Retrovirus-mediated gene delivery into male germ line stem cells // FEBS Lett. - 2000. - V.475(1). - P.7-10). The method includes injecting reporter gene lac Z directly in the testes and the subsequent determination of the transformation efficiency gene construct. Using PCR analysis, it was found that one of the 300 stem cell spermatogonia contained the transgene. The expression of reporter gene lacZ was observed in the testes of males over 6 months. The disadvantage of this method is that the transformation of the spermatogenic epithelium were used adult animals with a reduced number of stem cells spermatogonia that significantly worsened the result.

When creating the present invention, the task was to develop a method for genetic transformation of stem cells in the testes of rabbits in vivo using retrovirus-mediated gene transfer.

The technical result of the invention is achieved by a method of transformation of stem cells in the testes of rabbits in vivo, comprising injecting vector systems directly into the testes and the definition of EF is aktivnosti transformation gene constructs, characterized in that the transformation used 8-9-week-old male rabbits, which in the parenchyma of the testis injected vector design pX-RSVhGH encoding the human growth hormone, or pX-Ins encoding the human insulin, and the transformation efficiency determined using immunohistochemical set Novocastra (Sigma, USA).

The scheme used gene constructs shown in figure 1. While RSV - promoter of the rous sarcoma virus; SV - early promoter genes of SV40 virus; neo - neomycinphosphotransferase gene from transposon TP5 (where In - BamHI, E - EcoRI, S - Sad, SI - SalI, X - Xhol) (figure 1).

Example. Transformed stem cells spermatogonia on Historisch were identified by applying the following procedures.

Studied the morphology of the testes different age groups of male rabbits aged from 10 days to 6 months (with an interval of 7-14 days at slaughter or castration) by research hysteresis seminiferous tubules in order to identify age with a maximum number spermatogonia to conduct bioengineering manipulation.

On the basis of the obtained results was performed transfection of spermatogenic epithelium of rabbits in vivo in the age period of 8-9 weeks by multiple injection (5 to 8 injections per testis) gene constructs in the parenchyma of the testis. For genetic transformation of germ cells of the testes of rabbits in vio formed a group of animals depending on the gene construct (pX-Ins - the gene for human insulin under the control of the cytomegalovirus promoter and pX-RSVhGH gene of human growth hormone under the control of the promoter of rous sarcoma virus) and type of antiretroviral drug. For each of the used gene constructs were formed two experimental groups: 1-I - introduction cells packaging, 2-I - introduction the culture fluid (n=3 for each experimental group). Of packaging cells and culture fluid were injected with the number 1 million, respectively, and 0.5 ml (1×105) on the testis.

After 7 and 14 days after injection of animals with castration or slaughter samples were taken testes tissues for analysis of the presence of transformed cells. The expression of recombinant genes on tissue sections of the testes of rabbits was determined by immunohistochemical analysis using a set Novocastra (Sigma, USA). Performed analysis of 20 tissue sections (4-5 microns thick) with an interval of 50 μm and determined the frequency of genetic transformation gene constructs. Cells that synthesize the recombinant virus was stained a reddish-brown color.

The data processing was performed on a microscope (Opton" (Germany, lens X40, eyepiece h) using digital image processing (LLC System for microscopy and analysis").

The use of this method allowed us to show successful transformation of cells of the JV is mathoni when using both gene constructs.

The average frequency of integration of the gene construct containing the human growth hormone (number of transformed spermatogonia to the total number of spermatogonia transformed seminiferous tubules, expressed as a percentage) when using cell culture was 3.1±0,1%. The efficiency of genetic transformation of cells of the testes rabbit in vivo (the ratio of the number of transformed spermatogonia the total number on the cut) when using recombinant vector pX-RSVhGH on day 7 after injection, reaches 2.7·10-3.

The frequency of integration of the gene construct containing the gene for human insulin (pX-Ins), transformation of cells of the testes of rabbits in vivo was 3.5±0.1 and 3,4±0.1, respectively when using cell packaging and cultural liquid. The transformation efficiency of the cells of the testes of rabbits of the first group when using cells of packaging on the 7th day amounted to 2.3·10-3and was 3.1 times higher compared to the values obtained when the injection of the culture fluid. On day 14 after injection, the frequency of genetic transformation using cell packaging was 4.1·10-3that was 2.7 times higher compared to the use of supernatant.

Figure 2-3 shows the possibility of effective transformation of SCS rabbit when using the built structures pX-RSVhGH and pX-Ins (Fig.2-3). On figure 2 in the seminiferous tubule noticeable three spermatogonia transformed gene construct pX-RSVhGH (cells indicated by arrows). In the figure 3 in the seminiferous tubule presents two spermatogonia transformed gene construct pX-Ins (cells indicated by arrows). Paint hematoxylin-eosin. Magnification: lens X40, X10 eyepiece.

Thus, when using pX-RSVbGH transformed was one of 2,500 cells, while when using the pX-Ins is one of the 434 cells.

The obtained results confirm the possibility of a successful transformation of cells of the testes rabbit in vivo using retroviral gene constructs, while the efficiency of genetic transformation of cells of the testes of rabbits depends on the gene construct, the type of drug and the time after injection into the testes.

The proposed method can be used in experimental medicine and veterinary medicine, including genetic transformation of stem cells spermatogonia with the aim of obtaining transgenic animals.

The method of transformation of stem cells in the testes of rabbits in vivo, comprising injecting vector systems directly into the testes and the determination of the transformation efficiency of gene structures, characterized in that Thu is to transform the use 8-9-week-old male rabbits, which in the parenchyma of the testis injected vector design pX-RSVhGH encoding the human growth hormone, or pX-InS encoding the human insulin, and the transformation efficiency determined using immunohistochemical set Novocastra (Sigma, USA).



 

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