TransfectingSuspensionCells

實驗概要

將轉移基因整合到細胞染色體DNA上,形成穩定表達轉移基因的細胞系。

實驗原理

    細胞轉染技術是目前廣泛應用于病毒基因結構與功能以及基因調控等的研究。細胞轉染可分為短暫轉染和穩定(或永久) 轉染兩種。在短暫轉染中,被轉染基因并不整合至細胞染色體中,因而不能隨細胞分裂而傳代。轉入病毒基因的轉錄或復制可在DNA 導入后1～4 天通過收集被轉染的細胞進行分析。如果病毒基因整合至細胞染色體中則可隨細胞分裂而傳代,就能產生穩定或永久轉染的細胞系。

實驗步驟

1.        The  day before transfection, determine the number of cells needed for  transfection. For each 30 ml transfection, you will need 3 × 10⁷ cells  in 28 ml of FreeStyle? 293 Expression Medium. Expand cells accordingly,  taking into account the cell doubling time. For FreeStyle? 293-F cells,  this equates to passing cells at ~6–7 × 10⁵ cells/ml. 

2.        On  the day of transfection, determine the viability and the amount of cell  clumping from a small aliquot of cells using the trypan blue dye  exclusion method. Vigorously vortex for 45 seconds to break up cell  clumps and determine total cell counts using a Coulter Counter or a  hemacytometer. Viability of cells must be over 90%. 
Important:  For  best results, make sure to have a single cell suspension. It may be  necessary to vortex the cells vigorously for 10 to 30 seconds to break  up cell clumps. 

3.        Calculate the volume of cell suspension containing the number of cells needed for one transfection (you will need 3 × 10⁷cells for each 30 ml transfection). Place the shaker flask containing cells in a 37°C incubator on an orbital shaker. 

4.        For each transfection sample, prepare lipid-DNA complexes by performing
the following: 

1)        Dilute 30 μg of plasmid DNA in Opti-MEM? I to a total volume of 1 ml. Mix gently. 

2)        Dilute 60 μl of 293fectin? in Opti-MEM? I to a total volume of 1 ml. Mix gently and incubate for 5 minutes at room temperature. 
Note:  Longer incubation times may result in decreased activity. 

3)        After the 5 minute incubation, add the diluted DNA to the diluted 293fectin? to obtain a total volume of 2 ml. Mix gently. 

4)        Incubate for 20–30 minutes at room temperature to allow the DNA- 293fectin? complexes to form.

5.        While  the DNA-293fectin? complexes are incubating, remove the cell suspension  from the incubator and add  the appropriate volume of cell suspension  (see step 3) into a sterile, disposable 125 ml Erlenmeyer shaker flask.  Add fresh, pre-warmed FreeStyle? 293 Expression Medium to a total volume  of 28 ml for each 30 ml transfection. 

6.        After  the DNA-293fectin? incubation is complete, add the 2 ml of  DNA-293fectin? complex (from step 4) to each shaker flask containing the  cell suspension. To the negative control flask, add 2 ml of Opti-MEM? I  instead of DNA-293fectin? complex. Each flask should have a total  volume of 30 ml, and contain approximately 1 × 10⁶ viable cells per ml. 

7.        Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO₂ in air on an orbital shaker rotating at 125 rpm. 

8.        Harvest  cells or media (if recombinant protein is secreted) at approximately 48  hours post-transfection and assay for recombinant protein expression.