350 rub
Journal Technologies of Living Systems №5 for 2010 г.
Article in number:
FREQUENCY OF INTEGRATION OF A TRANSGENE AND VIABILITY OF THE RABBIT ZYGOTES, MICROINJECTION BY DIFFERENT GENNO-ENGINEERING DESIGNS
Keywords: gene engineering construction microinjection pronucleus transgenic rabbits
Authors:
S.I. Tevkin, Т.P. Trubicina, M.S. Shishimorova, V.А. Ezerskiy, V.P. Rjabyh
Abstract:
Nowadays, one of the most profitable directions of transgenic technology is generation of transgenic animals producing in their milk a biological activity substance for pharmaceutical proposes. The model of transgenic animals as a bioreactors bases on integration one of gene which code the therapeutic necessary protein. The expression this kind of genes is regulated by promoter sequence of various milk proteins such as S1-, β- and kappa-casein (Cn), β-lactoglobulin, β-lactoalbumin. Nevertheless received results are showed that frequency of integration transgene and level of expression of the recombinant proteins in the mammary gland are defined different factors: the specie of animal, the type of vector using for creation the gene construct, the size of recombinant DNA, the type of tissue specific promoter, the concentration of injecting DNA solution into the pronucleus and etc. The first and very important stage in technology of transgenesis is creation and research of gene-engineering constructs which can integrate into genom of mammalian embryos effectively. The testing these constructs on laboratory animals allow to choose more perspective for further using. The main purpose of our research was study the influence of microinjection gene-engineering constructs into pronucleus including the recombinant gene of human Lactoferrin (hLF) and human Granulocyte-colonystimulating factor (hG-CSF) under control regulatory elements gene of β-lactoglobulin Bos taurus on rabbit-s zygotes viability and study the efficacy of integration trans-gene in genom of the born puppies. After a microinjection in pronucleus gene-engineering con-structs BLg-hLf and BLg-hG-CSF in preimplantation rabbit-s zygotes stages of blastocyst has reached 47 and 80 %, accordingly, against 93 % in control group. The frequency of integration of the transgene defined at blastocyst stage after microinjection BLg-hLf construct was 10,4% and after microinjection BLg-hG-CSF was 4%, respectively. As a result of embryos transplantation to female-recipient after zygotes microinjection with recombinant gene of human Lactoferrin (hLF) under control regulatory elements gene of β-lactoglobulin Bos taurus transgenic offspring was received. Frequency of integration of a trans-gene after a recombinant construct injection (βLg-hLF) concerning newborn offspring has made 5% at general efficiency of trasgenesis  0,5%. The obtained data about presence of integration of a gene-engineering construct βLg-hLF in genome of the born offspring give us the basis for carrying out of the further researches on study-ing of a possible expression recombinant protein in milk and to studying of transgene transfer in number of generations. FREQUENCY OF INTEGRATION OF A TRANSGENE AND VIABILITY OF THE RABBIT ZYGOTES, MICROINJECTION BY DIFFERENT GENNO-ENGINEERING DESIGNS © Authors, 2009 S.I. Tevkin, Т.P. Trubicina, M.S. Shishimorova, V.А. Ezerskiy, V.P. Rjabyh Nowadays, one of the most profitable directions of transgenic technology is generation of transgenic animals producing in their milk a biological activity substance for pharmaceutical proposes. The model of transgenic animals as a bioreactors bases on integration one of gene which code the therapeutic necessary protein. The expression this kind of genes is regulated by promoter sequence of various milk proteins such as S1-, β- and kappa-casein (Cn), β-lactoglobulin, β-lactoalbumin. Nevertheless received results are showed that frequency of integration transgene and level of expression of the recombinant proteins in the mammary gland are defined different factors: the specie of animal, the type of vector using for creation the gene construct, the size of recombinant DNA, the type of tissue specific promoter, the concentration of injecting DNA solution into the pronucleus and etc. The first and very important stage in technology of transgenesis is creation and research of gene-engineering constructs which can integrate into genom of mammalian embryos effectively. The testing these constructs on laboratory animals allow to choose more perspective for further using. The main purpose of our research was study the influence of microinjection gene-engineering constructs into pronucleus including the recombinant gene of human Lactoferrin (hLF) and human Granulocyte-colonystimulating factor (hG-CSF) under control regulatory elements gene of β-lactoglobulin Bos taurus on rabbit-s zygotes viability and study the efficacy of integration trans-gene in genom of the born puppies. After a microinjection in pronucleus gene-engineering con-structs BLg-hLf and BLg-hG-CSF in preimplantation rabbit-s zygotes stages of blastocyst has reached 47 and 80 %, accordingly, against 93 % in control group. The frequency of integration of the transgene defined at blastocyst stage after microinjection BLg-hLf construct was 10,4% and after microinjection BLg-hG-CSF was 4%, respectively. As a result of embryos transplantation to female-recipient after zygotes microinjection with recombinant gene of human Lactoferrin (hLF) under control regulatory elements gene of β-lactoglobulin Bos taurus transgenic offspring was received. Frequency of integration of a trans-gene after a recombinant construct injection (βLg-hLF) concerning newborn offspring has made 5% at general efficiency of trasgenesis  0,5%. The obtained data about presence of integration of a gene-engineering construct βLg-hLF in genome of the born offspring give us the basis for carrying out of the further researches on study-ing of a possible expression recombinant protein in milk and to studying of transgene transfer in number of generations.
Pages: 42-49
References
  1. Езерский В.А., Иванова Л.Б., Шевченко В.Г. Создание генно-инженерной конструкции на основе регуляторных элементов гена b-лактоглобулина быка и структурного гена лактоферрина человека // Труды ВНИИФБИП. Боровск. 2006. Т. 45. С.121-137.
  2. Езерский В.А., Иванова Л.Б., Шевченко В.Г. Создание генно-инженерной конструкции, содержащей структурный ген G-CSF человека под контролем регуляторных элементов гена b-лактоглобулина крупного рогатого скота // Проблемы биологии продуктивных животных. Боровск. ВНИИФБиП. 2007. Т. 1. С. 123-131.
  3. Зиновьева Н.А.Получение и молекулярно-генетический анализ трансгенных кроликов с тканеспецифическими для молочной железы генными конструкциями: Автореф. дисс. на соиск. степени к.б.н. Дубровицы. 1994. 25 с.
  4. Козикова Л.В.Структурно-функциональная организация хромосом и изменение генома у трансгенных сельскохозяйственных животных: Автореф. дисс. на соиск. степени д.б.н. - Санкт-Петербург, 2005. - 35 с.
  5. Магда И.И.Оперативная хирургия. М.: Агропромиздат. 1990. С. 333.
  6. Сураева Н.М., Трухан Р.С. Некоторые методологические приемы введения чужеродного гена в зиготы животных // Генно-инженерные животные. 1995. Т. 1. С. 227-243.
  7. Bayna E.M., Rosen J.M. Tissue-specific, high level expression of the rat whey acidic protein gene in transgenic mice // Nucl. Acids Res. 1990.
    V. 18(10). P. 2977-2985.
  8. Bijovet A.G., Kroos M.A., Pieper F.R. Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GCD II mice // Hum. Med. Genet. 1998. V. 7. P. 1815-1824.
  9. Brem G., Besenfelder U., Zinovieva N. Mammary gland specific expression of chymosin constructs in transgenic rabbits // Theriogenology. 1995. V. 43(1). V. 175-175.
  10. Brinster R.L., Allen I.M. Factors affecting the efficience of introducing foreing DNA into mice by microinjecting eggs // Proc. Natl. Acad. Sci. USA. 1985. V. 82. P. 4438-4442.
  11. Blin N., Stafford D.V. // Nucl. Acids Res. 1976. V. 3. P. 2303 - 2308.
  12. Ebert K.M., Selgrath J.P., DiTullio P. Transgenic production of a variant of human tissue-type plasminigen activator in goat milk: generation of transgenic goats and analysis of expression // Bio/Technol. 1991. V. 9. P. 835 - 838.
  13. Hammer R.E., Pursel V.G., Rcxroad C.E.Jr., Wall R.J., Bolt D.J., Ebert K.M., Palmiter R.D. and Brinster R.I. Production of Iransgenic rabbits, sheep and pigs by microinjection // Nature. 1985. V. 315. P. 680 - 683.
  14. Henninghausen L. The mammary gland as a bioreactor: Production of foreign protein in milk // Protein expression and purification. 1990. V. 1. P. 3 - 8.
  15. Hirabayashi M., Ilirao M., Iloehi S. Effects of superovulatory protocols on the recovery and subsequent developmental potential of rabbit zygote // J. Exp. Anim. Techno. 2000. V. 35. P. 23 - 28.
  16. Hirabayashi M, Takahashi R, Ttod K. Comparative Study on the Integration of Exogenous DNA into Mouse, Rat, Rabbit and Pig Genomes // Exp. Anim. 2001. V. 50(2). P. 125 - 131.
  17. Krimpenfort P., Rademakers A., Eyestone W. Generation of transgenic dairy cattle using «in vitro» embryo production // Bio.Technology. 1991. V. 9. P. 844 - 847.
  18. Kim S.J., Lee K.-W., Yu D.-Y. Expression analysis of a bovine В-casein human lactoferrin hybrid gene in transgenic mice // J. Reprod. Dev. 1997. V. 43. P. 143 - 149.
  19. Korhonen V.P., Tolvanen M., Hyttinen J.M., Uusi-Oukari M., Sinervirta R., Alhonen L., Jauhiainen M., Janne O.A., Janne J. Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits // Eur. J. Biochem. 1997. V. 245(2). P. 482 - 489.
  20. Lonnerdal B. and Lyer S. Lactoferrin: molecular structure and biological function // Annu. Rev. Nutr. 1995. V. 15. P. 93 - 110.
  21. Lieshke G.J., et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor deficiency, and impaired neutrophil mobilization // Blood. 1994. V. 84. P. 1737-1746.
  22. Limonta J.M., Castro F.O., Martinez R. Transgenic rabbits as bioreactors for the production of human growth hormone // J. Biotechnology. 1995. V. 40. P. 49 - 58.
  23. Nottle M. B., Haskard K. A., Verma P. J., et.al. Effect of DNA concentration on transgenesis rates in mice and pigs // Transgenic Res. 2001. V. 10(6). P. 523-31.
  24. Riego E., Limonta J., Aguilar A., Perez A., Armas R.de, Solano R., Ramos B., Castro F.O. and de la
    Fuente J.     Prodaction of transgenic mice and rabbits that carry and exspress the human tissue plasminogen activator cDNA under the control of a Bovine alpha SI casein promoter // Theriogenology. 1993. V. 39. P. 1173-1185.
  25. Sokolov A. V. A study of recombinant human laktoferrin secreted in milk of transgenic mice // Doklady Biochemistry and Biophysics. 2006. V. 411. P. 336 - 338.