File:PDNA transfer via PepFect14 (close-up).jpg
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Eesti: CHO rakku tuuma on viidud plasmiidset DNA-d (pGL3; 4,7 kDa) kasutades selleks PepFect14 rakku sisenevaid peptiide. Vasakul äärel on näha rakutuum, paremal mitokondrid. Mõõtlõik on antud pikkusega 500 nm. Transmissioonelektronmikroskoopia. Tartu Ülikooli molekulaar- ja rakubioloogia instituut. [1]
Plasmiidse DNA puhul moodustati esmalt pDNA ja streptavidiin-nanokuld (SA-NG) kompleksid, et pDNA oleks elektronmikroskoobis paremini detekteeritav. Selleks inkubeeriti eelnevalt biotinüleeritud pDNA molekule SA-NG konjugaatidega molekulaarsel suhtel 1:3 (pDNA:SANG) 30 min toatemperatuuril. Seejärel inkubeeriti pDNA-SA-NG komplekse PepFect14-ga laengusuhtel 1:2 30 min toatemperatuuril. Seejärel lisati kompleksidele 900 μl söödet, saavutades lõppruumala 1 ml. Vahetult enne komplekside rakkudele kandmist pesti neid kaks korda söötmega ning seejärel kanti rakkudele peale inkubatsioonilahus. Rakke inkubeeriti 37°C juures 4 h. Rakud fikseeriti glutaaraldehüüdiga 0,2 M Na-kakodülaatpuhvris (pH 7,4). Suurendamaks kullamärgist (1,4 nm) hõbetati rakke saavutades nõnda märgiste läbimõõt ~10–15 nm. Rakud on kontrasteeritud osmiumtetroksiidiga.
English: Transmission electron micrograph of a CHO cell, that has plasmid DNA transferred into its nucleus with PepFect14 cell-penetrating peptides. On the right side of the image there are mitochondria. pDNA (pGL3; 4,7 kDa) has been marked with nanogold, that has been enlarged with silver coating to make it visible under the microscope. The bar in the right indicates a dictance of 500 nm. Institute of Molecular and Cell Biology, University of Tartu. |
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| Source | Own work |
| Author | Ivo Kruusamägi |
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