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FREQUENCY OF INTEGRATION OF A TRANSGENE AND VIABILITY OF THE RABBIT ZYGOTES, MICROINJECTION BY DIFFERENT GENNO-ENGINEERING DESIGNS

Keywords:

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. 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.
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