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Transgene expression in plant cell

100%

Niemirowicz-Szczytt K., Bartoszewski G.

Biotechnologia

2000

issue 4

11-23

Transgene incorporation in plant genome does not mean that the gene will be active and will show expression at the required level. It is often the case that transgenic plants do not exhibit the activity of the gene introduced. Gene expression is known to be dependent on many factors such as: chimeric gene structure, proper and stable integration to the genome and proper transcription and translation. The knowledge of transgene regulation is essential for the understanding of the transcription and translation mechanisms. Each plazmid component is vital for later transgene expression. This refers to the size of T-DNA introduced, location of particular elements, leader sequences length, AUG sequences, introns presence, optimum coding gene synthesis, removal of RNA instability signals and many other possible modifications.

Expressive gene structures of proper and modified genes

88%

Lipinski D., Szalata M., Kalak R., Plawski A., Nuc K., Kala M., Juzwa W., Slomska K., Gronek P., Jura J., Smorag Z., Pienkowski M., Slomski R.

Biotechnologia

2003

issue 1

48-73

The genetic construct WAP 6xHisHGH containing the gene encoding human growth hormone (hGH) and WAP promoter expressed in mammary gland of animals was prepared. The 5? end of the gene was modified by the addition of sequence encoding six histidine residues and the sequence recognized by thrombin. In this way, the growth hormone can be easily purified by affinity chromatography and cleaved with thrombin to an active form. In the next step, the genetic construct was introduced by microinjection into male pronuclei of fertilized oocytes. Transgene was detected in male rabbit of F0 generation (number 61). Twelve offspring of founder rabbit of generation F1 indicated transgene sequences. The presence of growth hormone was revealed in the samples of milk accumulated during the lactation of females of F1 generation. The genetic constructs containing chain 1 and chain 2 of Feld1, and the major allergen produced by cat (Fedlis domesticus) were prepared. Both genes were inactivated by introduction into the sequences a positive selectable marker aminoglycoside phosphotransferase (resistant to neomycin). Outside the region of homology to Feld1 chain 1 and chain 2 genes, the negative selectable marker ? thymidine kinase gene was introduced. The genetic constructs pNTKFd1 and pNTKFd2 can be used in further experiments involving the inactivation of Feld1 genes in cat cells. Both genes were modified by site-directed mutagenesis using megastarter with Stop codon for premature termination of translation. The presence of mutation was confirmed by sequencing. The genetic constructs with human hGH gene and cat Feld1 gene were introduced into the bovine and cat fetal fibroblasts respectively in co-transfection with plasmid pGT-N29 containing positive selectable marker by lipofection, precipitation and electroinjection methods. After the selection, surviving cells were subjected to further molecular analysis. The stabile incorporation of the genetic constructs WAP 6xHisHGH and WAPHGH into the genome were observed.

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2 Biotechnologia

1 2003

1 2000

1 Bartoszewski G.

1 Gronek P.

1 Jura J.

1 Juzwa W.

1 Kala M.

1 Kalak R.

1 Lipinski D.

1 Niemirowicz-Szczytt K.

1 Nuc K.

1 Pienkowski M.

1 Plawski A.

1 Slomska K.

1 Slomski R.

1 Smorag Z.

1 Szalata M.

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Transgene expression in plant cell

Expressive gene structures of proper and modified genes