Fandom

VroniPlag Wiki

Vpr/Fragment 033 01

< Vpr

31.268Seiten in
diesem Wiki
Seite hinzufügen
Diskussion0 Share

Störung durch Adblocker erkannt!


Wikia ist eine gebührenfreie Seite, die sich durch Werbung finanziert. Benutzer, die Adblocker einsetzen, haben eine modifizierte Ansicht der Seite.

Wikia ist nicht verfügbar, wenn du weitere Modifikationen in dem Adblocker-Programm gemacht hast. Wenn du sie entfernst, dann wird die Seite ohne Probleme geladen.


Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 33, Zeilen: 1 ff. (cpl.)
Quelle: Fontanari Krause 2006
Seite(n): 91 f., Zeilen: 91: 8ff - 92: 1 -4
3.4 Retroviral infection of primary BM cells.

Primary mouse bone marrow (BM) cells were transduced as previously described (Pineault et. al., 2003a). Briefly, BM cells were obtained by flushing both femurs and tibias of donor mice treated 4 days previously with 150 mg/kg 5-fluorouracil injected into the tail vein. 5-flourouracil eliminates cycling cells from the animal, thus enriching the bone marrow for primitive hematopoietic progenitors, which are non-cycling or quiescent in nature (Reya et. al., 2001). Cells were stimulated for 48 hrs in DMEM supplemented with 15% FBS, 10 ng/ml mIL-6, 6 ng/ml mIL-3 and 100 ng/ml murine stem cell factor (SCF). For transduction, cells were co-cultured for 48h with irradiated (40 Gy) GP+E86 virus producing cells in the same medium with an addition of 5 μg/ml protamine sulfate with ETV6/CDX2/GFP or Cdx2/YFP GP+E86 producers or with a mixture of 40 to 50% CDX2/YFP and 50 to 60% ETV6/CDX2/GFP producers in co-transduction experiments. Protamine sulfate prevents aggregation of viral particles, thus increasing efficiency of transduction. Loosely adherent and nonadherent BM cells were harvested from the co-culture 48h post transduction, BM cells were furtherer cultured in fresh medium with cytokine cocktail for 48h to allow expression of EGFP or EYFP. Transduced BM cells were sorted out by florescence activated cell sorting (FACS) using EGFP or EYFP as a marker.

In Vitro Assays

3.5 Proliferation Assay.

To study the proliferative potential in vitro of BM cells transduced with ETV6/CDX2, Cdx2, its mutants and ETV6/CDX2 + Cdx2, we performed a proliferation assay by plating an equal number of successfully transduced bone marrow cells directly after sorting in DMEM supplemented with 15% FBS, 10 ng/ml mIL-6, 6 ng/ml mIL-3 and 100 ng/ml mSCF (standard medium) (Tebubio GmbH, Offenbach, Germany) at 370C in a humidified CO2 incubator. The [cells were subjected to half-media change every 7 days and their proliferation assessed on the same day by counting viable cells after trypan exclusion.]

[page 91]

5.10.3. Retroviral infection of primary BM cells

Primary mouse bone marrow (BM) cells were transduced as previously described (Pineault et al., 2003). Briefly, BM cells were obtained by flushing both femurs and tibias of donor mice treated 4 days previously with 150 mg/kg 5-fluorouracil (myeloid condition) injected into the tail vein. Mice without 5-fluorouracil treatment (for lymphoid condition) were also used in this work. 5-flourouracil eliminates cycling cells hematopoietic cells from the animal, thus enriching the bone marrow for primitive hematopoietic progenitors, which are non-cycling or quiescent in nature (Reya, 2001). Cells were stimulated for 48 hrs in DMEM supplemented with 15% FBS, 10 ng/ml IL6, 6 ng/ml IL3 and 10 ng/ml mSCF.

For lymphoid condition assays, cells were stimulated for 24 h in DMEM supplemented with 15% FBS, 10 ng/ml IL7, 6 ng/ml mutant FLT3 and 10 ng/ml mSCF.

For transduction, cells were co-cultured for 48h with irradiated (40 Gy) GP+E86 virus producing cells in the same medium with the addition of 5 μg/ml protamine sulfate. Protamine sulfate prevents aggregation of viral particles, thus increasing efficiency of transduction. Loosely adherent and non-adherent BM cells were harvested from the coculture 48h post transduction, BM cells were furtherer cultured in fresh medium with cytokine cocktail for 48 h or 24 h (myeloid or lymphoid condition) to allow expression of EGFP. Transduced BM cells were sorted by fluorescence activated cell sorting (FACS) using EGFP as a marker.

5.10.4. In Vitro Assays

5.10.4.1. Proliferation Assay

To study the proliferative potential in vitro of BM cells transduced with OSTL expressing retroviruses, a proliferation assay was performed by plating successfully transduced bone marrow cells directly after sorting in DMEM supplemented with 15% FBS, 10 ng/ml mIL-

[page 92]

6, 6 ng/ml mIL-3 and 100 ng/ml murine stem cell factor (SCF) (standard medium) (Tebubio GmbH, Offenbach, Germany) at 37°C in a humidified 5% CO2 incubator. The cells were subjected to half-media change every 7 days and their proliferation assessed on the day of media change by counting viable cells after trypan exclusion.

Anmerkungen

The source is not mentioned here.

Sichter
(SleepyHollow02) Schumann

Auch bei Fandom

Zufälliges Wiki