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Сельскохозяйственная биология, 2010, № 6, с. 16-19.

УДК [636.4+636.5]:631.523:577.212:57.088

STUDY OF FACTORS AFFECTED THE EFFICIENCY OF GENE TRANSFER INTO THE MALE GERM CELLS OF AGRICULTURAL ANIMALS

N.A. Volkova¹, N.A. Zinovieva¹, L.A. Volkova¹, N.S. Lotsmanova¹, L.K. Ernst³

The factors affected the efficiency of the address DNA transfer into the chicken and pigs spermatogonia cells in vivo were evaluated. The maximal efficiency of gene transfer was observed using suspension of packaging cells containing the retrovirus vector pX-Ins in concentration of 1 and 5 million cells per testis for chicken and pigs, respectively.

Key words: cell engineering, transgenesis, retrovirus vectors, pigs, chickens.

 

Development of genetic modification techniques based on using germ cells as vectors for targeted gene transfer is considered to be the alternative to traditional method for producing transgenic animals by DNA microinjection into the pronucleus of zygotes (1-4). This issue is the subject of interest primarily owing to the natural ability of spermatogenic epitheliocytes to absorb heterogeneous DNA (hDNA) and transfer it into the egg-cell during fertilization. Such hDNA integrated into the genome can be stably inherited by further generations, while reducing time and money costs for obtaining transgenic animals because all manipulations are performed upon adult individuals.

Transformation of germ cells can be done by different methods (lipofection, electroporation and retroviral transduction) (5-7). However, the systems providing efficient transferring of recombinant DNA into spermatogonia cells still haven’t been developed.

In this context, the authors studied the factors affecting the efficiency of genetic transformation of spermatogonia using retroviral vectors.

Technique. For transformation of target cells, the retroviral vector pX containing cDNA of human growth hormone (gene construct pX-RSVhgh) and cDNA of human insulin (gene construct pX-Ins) was used. For packaging of retroviral vectors, the line of packaging cells GP + envAm12 was applied. The gene constructs were kindly provided by Candidate Biol. Sci. I.K. Fomin (Institute of Genetics and Cytology of the NAS of Belarus, Minsk).

The source of gene constructs were packaging cells producing recombinant retrovirus (vector pX) and the viral preparation obtained by collecting the growth medium (DMEM-based) used for cultivation of packaging cell.

Firstly, transformation of spermatogenic epithelium cells of boars and chickens by the gene constructs pX-RSVhGH and pX-Ins was performed in vitro culture in order to compare their effectiveness at genetic modification of selected target cells. The frequency of genetic transformation was defined as the percentage ratio: the number of resulting genetically transformed clones to the total number of infected cells.

The next stage of work was introduction of the vectors into the testis parenchyma of experimental animals in vivo by multiple injections (5-8 per testis). Two experiments were carried out. In the first experiment (two groups of boars and two groups of roosters, 3 individuals in each group), both constructs (pX-RSVhGH and pX-Ins) were administered as a viral preparation in order to compare the efficiency of cell transformation by each of these constructs. The most efficient construct revealed by these tests was supposed to be used in further work. In the second experiment (three groups of boars and three groups of roosters, 3 individuals in each group), the quantity of injected gene construct providing increased efficiency of transgenesis was determined. The source of gene construct was the suspension of packaging cells (at different doses) in DMEM medium; mitosis in these cells was preliminary blocked with mitomycin C.

To detect the expression of retroviral vectors, histological preparations of testes obtained at castration or slaughter of animals were made (8). Immunohistochemical staining of sections was performed using specific antibodies to recombinant proteins. A chromogen - diaminobenzidine (DAB). For the analysis, at least 20 histological sections 4-5 microns thick were cut at an interval of 50 microns.

The obtained data were statistically processed (9).

Results. The frequency of genetic transformation of spermatogonia of boars and chickens in vitro varied depending on transfection method and the used gene construct. The highest number of genetically transformed clones was obtained by co-cultivation with packaging cells. In particular, the vector pX-Ins provided from 0,4 to 0,6% transformed cells depending on the species. The infection of spermatogenic epitheliocytes with viral preparation showed 2,2-3,1 times lower efficiency of  transformation of target cells.

Similar results were obtained when using the retroviral vector pX-RSVhGH. In this case, the efficiency of genetic transformation in primary culture of testis cells was slightly lower compared to the gene construct pX-Ins: in the variant with packaging cells, it amounted to 0,2-0,5%, and when infection with viral preparation - 0 ,1-0, 2%.

The results of experiments in vitro were the basis for next research - the transferring of recombinant DNA into the cells of spermatogenic epithelium of boars and chickens in vivo.

The comparison of effectiveness of the vectors pX-RSVhGH and pX-Ins injected into the testes of boars and roosters showed that in the variant with pX-RSVhGH, integration and expression of the transgene in spermatogenic epithelium cells was observed only in chickens: positive signals were found in 10 of 60 analyzed sections (Table 1).

The number of transformed tubules per one section varied from 0,22 ± 0,04 to 0,42 ± 0,11, and averaged 0,05 ± 0,09 over the group, while the efficiency of transformation equaled to 0,06% (percentage ratio: the number of transformed spermatogonia to their total number in all investigated seminiferous tubules). The maximum number of transformed cells didn’t exceed 3 per one seminiferous tubule.

In boars, the vector pX-RSVhGH didn’t provide the formation of transgenic tubules and spermatogonia.
The introduction of pX-Ins was more effective for both chickens and boars: in all experimental animals, this construct was found to be integrated and expressed in spermatogonia. Immunohistochemical tests revealed the presence of transformed cells in 9-12 of 60 analyzed sections of the testes, the number of transformed cells per one seminiferous tubule varied from 1 to 2 at total per section 4. The efficiency of transformation of spermatogonia reached 0,08%.

The suspension of packaging cells as a source of the vector pX-Ins provided higher efficiency of transgenesis. Increased doses of injected cells resulted in raised efficiency of transformation (Table 2). In particular, for boars, this parameter was 0,40% at the dose of 3 million cells/testis and increased by 0,49%  when 5 million cells/testes were injected. In chickens, at the doses of packaging cells 0,5 million and 1 million cells per testis, this indicator amounted to, respectively, 0,48 and 0,66%.

At the same time, it should be noted a slight decrease in efficiency of transformation of spermatogonia with increasing the doses of injected vectors per testis (from 5 million to 7 million cells in boars and from 1 million to 2 million cells in chickens). This fact may be associated with immune response to the presence of heterogeneous cellular material, while the smaller doses of introduced gene constructs (up to 5 million cells in boars and 1 million cells in chickens) caused less pronounced response of the organism. Therefore, the doses of packaging cells (per testis) equal to 5 million for boars and 1 million for roosters should be considered the optimum source of retroviral vectors in terms of efficient transformation of spermatogonia.

Thus, the obtained results suggest the possibility of using retroviral vectors for genetic transformation of germ cells of boars and roosters, as well as the prospects for using this system of gene transfer aimed at producing transgenic animals with desired properties.

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1All-Russia Research and Development Institute of Livestock Husbandry, RAAS, Moscow province, Podolsk region, Dubrovitsy settlement 142132, Russia e-mail: natavolkova@inbox.ru; 2Russian Academy of Agricultural Sciences, Moscow 117218, Russia, e-mail: ernstrashn@yandex.ru

Received September 27, 2010

All-Russian Institute for Selection and Seed-Breeding of Vegetables All-Russian Selection and Technological Institute of Horticulture and Breeding Nursery Institute of Agricultural Biotechnology Joint Stock Company "GosNIISyntezBelok"