Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2015, V. 50, № 4, pp. 458-466.

UDC 636.52/.58:573.6.086.83:636.082

doi: 10.15389/agrobiology.2015.4.458eng

THE STUDY OF FACTORS AFFECTED THE GENE TRANSFER EFFICIENCY IN CHICKEN EMBRYONIC CELLS BY APPLICATION
OF LENTIVIRAL VECTORS

N.A. Volkova1, I.K. Fomin1, E.K. Tomgorova1, A.N. Vetokh1,
E.R. Mennibaeva1, G. Brem2, N.A. Zinovieva1

1L.K. Ernst All-Russian Research Institute of Animal Husbandry, Federal Agency of Scientific Organizations, pos. Dubrovitsy, Podolsk Region, Moscow Province, 142132 Russia, e-mail natavolkova@inbox.ru;
2Institute of Animal Breeding and Genetics, VMU, Veterinärplatz, A-1210, Vienna, Austria, e-mail gottfri-ed.brem@agrobiogen.de

Supported financially by Federal Agency of Scientific Organizations (the State Registration Number NIR 01201455101).

Received January 29, 2015

 

Lentivirus-mediated gene transfer is being the one of the attractive method for genetic modification of chicken (S.C. Chapman et al., 2005; C.A. Smith et al., 2009; N.A. Volkova et al., 2013). However, the efficiency of thansgenesis of the chicken embryonic cells has been shown to be relatively low. Therefore, a large number (60,000 to 100,000) of embryonic cells at the start of incubation and the virus preparations with high titers (about 109 particles per milliliter) remain one of a crucial problem the researchers are facing with when try to achieve a satisfactory transgene introduction. The aim of the present study was to determine the optimal conditions for production and application of the modified lentiviral vector system of second generation for the transgenesis of chicken embryos. The vector system consisted of three different plasmids: psPAX2, containing gag-pol genes; pLPG, coding envelop glycoprotein G of vesicular stomatitis virus (VVC-G) and pWPXL, the self-inactivated lentiviral vector, carrying eGFP (enhanced green fluorescence protein) gene under control of promoter region of the human elongation factor 1 alpha-encoding gene. To produce the recombinant virus particles and to determine the virus titers we used human cell line 293T. The injections of the virus preparations into the chicken embryos were performed at the different stages: from 20 to 24 hours (group 1) and from 50 to 55 hours (group 2) of incubation. To detect the transgenesis efficiency and the number of the integrated copies of the transgene the total DNA was extracted from embryos on the day 7 of incubation and analyzed for the presence of specific eGFP sequences by real-time PCR. The maximal titers of the virus preparations were produced by the ratio of the psPAX2, pLPG and pWCAG plasmids equal 1:1:3 and were 2.4x107 CFU/ml before ultracentrifugation and 6.2x108 CFU/ml after concentration by ultracentrifugation. The efficiency of genetic transformation which was evaluated as the part of the transformed cells from the overall number of analyzed cells was 78.0 and 31.0 % in the groups 1 and 2, respectively. It was shown, that the alteration in the ratios between components of the vector system comparing to the standard scheme allows significantly increase the titers of the produced virus preparations. The biological titers of the virus preparations of 108 CFU/ml are sufficient to infect up to eighty percent of cells at the earlier stages of embryo development. Presumably, at earlier embryogenesis the cells were infected with more viral particles resulting in different number of the transgene copies integrated into cell genome in the groups 1 and 2. From the obtained results, the efficiency of transgenesis by means of the lentiviral vectors ranged from 30.0 to 34.3 % and varied slightly depending on time after the embryos incubation began. These results indicate that only a part of embryonic cells is usually available for viral infection. Injection of embryos at different intervals of incubation by viral preparations with similar titers produced the populations of embryonic cells with different amounts of vector copies in the cell genome. So the efficiency of hen embryo thransgenesis does not depend on the stage of its development at least for 55 hours of the embryogenesis and can be predictable.

Keywords: lentiviral vectors, molecular cloning, transfection, transgenic animals.

 

Full article (Rus)

Full text (Eng)

 

REFERENCES

  1. Mizuarai S., Ono K., Yamaguchi K., Nishijima K-I., Kamihira M., Iijima S. Production of transgenic quails with high frequency of germ-line transmission using VSV-G pseudotyped retroviral vector. Biochem. Biophys. Res. Commun., 2001, 286: 456-463 CrossRef
  2. McGrew M.J., Sherman A., Ellard F.M., Lillico S.G., Gilhooley H.J., Kingsman A.J., Mitrophanous K.A., Sang H. Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep., 2004, 5: 728-733 CrossRef
  3. Chapman S.C., Lawson A., Macarthur W.C., Wiese R.J., Loechel R.H., Burgos-Trinidad M., Wakefield J.K., Ramabhadran R., Mauch T.J., Schoenwolf G.C. Ubiquitous GFP expression in transgenic chickens using a lentiviral vector. Development, 2005, 132: 935-940 CrossRef
  4. Smith C.A., Roeszler K.N., Sinclair A.H. Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector, RCASBP. Differentiation, 2009, 77(5): 473-482 CrossRef
  5. Volkova N.A., Volkova L.A., Fomin I.K., Zinov'eva N.A., Gorelik L.Sh., Lotsmanova N.S. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2013, 2: 58-61 CrossRef, CrossRef
  6. Volkova N.A., Tomgorova E.K., Bagirov V.A., Beloglazov D.V., Zinov'e-
    va N.A., Volkova L.A., Ernst L.K. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2009, 6: 44-48.
  7. Kamihira M., Ono K., Esaka K., Nishijima K., Kigaku R., Komatsu H., Yamashita T., Kyogoku K., Iijima S. High-level expression of single-chain Fv-Fc fusion protein in serum and egg white of genetically manipulated chickens by using a retroviral vector. J. Virol., 2005, 79(17): 10864-10874 CrossRef
  8. Scott B.B., Velho T.A., Sim S., Lois C. Applications of avian transgenesis. ILAR J., 2010, 51(4): 353-361 CrossRef
  9. Furlan-Magaril M., Rebollar E., Guerrero G., Fernandez A., Moltaі E., Gonzalez-Buenda E., Cantero M., Montoliu L., Recillas-Targa F. An insulator embedded in the chicken β-globin locus regulates chromatin domain configuration and differential gene expression. Nucl. Acids Res., 2011, 39(1): 89-103 CrossRef
  10. Scott B.B., Lois C. Generation of tissue-specific transgenic birds with lentiviral vectors. PNAS, 2005, 102(45): 16443-16447 CrossRef
  11. Dougherty D.C., Sanders M.M. Estrogen action: revitalization of the chick oviduct model. Trends Endocrinol. Metab., 2005, 16: 414-419 CrossRef
  12. Shimizu M., Losos J.K., Gibbins A.M. Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes. Biochem. Cell Biol., 2005, 83(1): 49-60 CrossRef
  13. Lillico S.G., Sherman A., McGrew M.J., Robertson C.D., Smith J., Hasl-
    am C., Barnard P., Radcliffe P.A., Mitrophanous K.A., Elliot E.A., Sang H.M. Oviduct-specific expression of two therapeutic proteins in transgenic hens. PNAS, 2007, 104(6): 1771-1776 CrossRef
  14. Byun S.J., Kim S.W., Kim K.W., Kim J.S., Hwang I.S., Chung H.K., Kan I.S., Jeon I.S., Chang W.K., Park S.B., Yoo J.G. Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens. Biosci. Biotechnol. Biochem., 2011, 75(4): 646-649 CrossRef
  15. Schomber T., Kalberer C.P., Wodnar-Filipowicz A., Skoda R.C. Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells. Blood, 2004, 103(12): 4511-4513 CrossRef
  16. Sambrook J., Fritsch E.F., Maniatis T. Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor, NY, 1989.
  17. Tiscornia G., Singer O., Verma I.M. Production and purification of lentiviral vectors. Nat. Protoc., 2006, 1(1): 241-245 CrossRef

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