Team:Heidelberg/Project/Mouse Infection
From 2010.igem.org
Laura Nadine (Talk | contribs) (→virus production) |
Laura Nadine (Talk | contribs) |
||
Line 24: | Line 24: | ||
(4) The on-targeting construct consisted of two independent viruses which were co-infected into mice, as well. One of these viruses packaged the Tet Repressor (TetR) driven by an SV40 promoter. The expression of TetR is under the control of miR-122 as four binding sites of this miRNA were cloned into the 3’UTR of the gene. The second virus was composed of an sv40 promoter driving the Tet operator (TetO2) which monitors the expression of luc2. With this setup, luc2 expression should be inhibited by the TetR in all mice tissues except for liver cells, where TetR is downregulated by miRNA 122. | (4) The on-targeting construct consisted of two independent viruses which were co-infected into mice, as well. One of these viruses packaged the Tet Repressor (TetR) driven by an SV40 promoter. The expression of TetR is under the control of miR-122 as four binding sites of this miRNA were cloned into the 3’UTR of the gene. The second virus was composed of an sv40 promoter driving the Tet operator (TetO2) which monitors the expression of luc2. With this setup, luc2 expression should be inhibited by the TetR in all mice tissues except for liver cells, where TetR is downregulated by miRNA 122. | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
===mice injection=== | ===mice injection=== | ||
Line 43: | Line 37: | ||
===contructs=== | ===contructs=== | ||
===production of recombinant virus=== | ===production of recombinant virus=== | ||
+ | The viruses were produced on HEK 293-T cells and purified on an iodixanol gradient according to [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Virus_Production protocol]. | ||
+ | |||
+ | Before infection, the titer of the viruses was quantified using .[https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Quantitative_Realtime_PCR quantitative realtime PCR] | ||
+ | |||
===procedure involving animals=== | ===procedure involving animals=== | ||
The mouse experiments were conducted in accordance with the animal facility of the german cancer research institute of Heidelberg. Female NMRI mice were purchased from....??. At 8-10 weeks of age the animals were injected in the tail vein (TV), with ~1x10^11 particles of AAV-SV40-luciferase in 200µl of 1x phosphate-buffered saline. The mice are transferred to a holding device which restrains the mouse while allowing access to the tail vein. The tails were warmed before the injections and injections were carried out usind 27 gauge needles. All the mice recoverd from the injection quickly without loss of mobility or interruption of grooming activity. | The mouse experiments were conducted in accordance with the animal facility of the german cancer research institute of Heidelberg. Female NMRI mice were purchased from....??. At 8-10 weeks of age the animals were injected in the tail vein (TV), with ~1x10^11 particles of AAV-SV40-luciferase in 200µl of 1x phosphate-buffered saline. The mice are transferred to a holding device which restrains the mouse while allowing access to the tail vein. The tails were warmed before the injections and injections were carried out usind 27 gauge needles. All the mice recoverd from the injection quickly without loss of mobility or interruption of grooming activity. |
Revision as of 13:53, 25 October 2010
|
|
|||