Evaluación del resumen del proyecto

4.3.3.1. Order of mixing

Changing the order in which the components of non- viral transfection complexes are mixed has been demonstrated to effect the efficiency of transfection (Cheng, 1996). Transfection complexes were prepared with

peptide 1 (I), pGL2 plasmid DNA (D) and Lipofectin (L) mixed in all the possible orders (Figure 4.12). 400000-, c 5 30000 0- o &_ Q. U) 2 2000004 ÔC 1000004 A

0 LID LDI OIL DU IDL ILD

Order of mixing of complex

F ig u r e 4 .1 2 T r a n s f e c t io n o f n o r m a l f ib r o b la s t s w ith t r a n s f e c t io n c o m p le x e s fo r m e d b y m ix in g th e c o m p o n e n t s in d iff e r e n t w a y s .

L= Lipofectin, 1= Integrin targeting peptide, D= DNA therefore ILD indicates that I was added to the container first then L then D, with mixing after each stage.

Vector: Lipofectin- peptide 1 - luciferase plasmid (optimal weight ratio).

Transfection time: 6h Expression time: 68h

Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells.

The order of mixing the transfection complex components altered the expression levels greatly. Addition of peptide to Lipofectin followed by the DNA gave the highest expression level. Since LI is different from IL there may be some interaction between the components and the plastic container.

Chapter 4. Optimization of vector system

4.3.3.2. Incubation of Lipofectin and peptide before addition of plasmid DNA

The effect of mixing Lipofectin and peptide and pre- incubating at room temperature before adding the DNA and incubating the complex was examined (Figure 4.13). 400000n c 5 3 0000 0- o g ' 2000004 ^ 1000004

Control 0 min 30 min

Incubation time of lipofectin and peptide before addition of DNA

F ig u r e 4 .1 3 T r a n s f e c tio n o f n o r m a l f ib r o b la s t s w it h t r a n s fe c tio n c o m p le x e s w h ic h h a d b e e n p r e p a r e d b y f ir s t m ix in g L ip o fe c tin w ith p e p tid e a n d a d d in g th e p la s m id D N A e it h e r im m e d ia t e ly o r a ft e r 3 0 m in .

Vector: Lipofectin- peptide 1 - luciferase plasmid (optimal weight ratio).

Transfection time: 6h Expression time: 68h

Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells,

Pre- incubation of Lipofectin and peptide before the addition of the DNA decreased the luciferase expression. This pre- incubation may increase the interaction of the complex components with the container.

Chapter 4._________________________________________________ Optimization of vector system

4.3.3.3. Transfection complex formation time

The effect of altering the time for complex formation was analysed (Figure.4.14). 50000 0- Ç 400000 'S o cL 300000 3 2000001 0^ lOOOOOn

Control 15 min 30 min 1h 2h 4h 6h

Complex incubation time

F ig u r e 4 .1 4 T r a n s f e c tio n o f n o r m a l f ib r o b la s t s w it h t r a n s f e c tio n c o m p le x e s w h ic h h a d b e e n in c u b a t e d f o r d if f e r e n t tim e s b e tw e e n m ix in g o f th e t r a n s f e c t io n c o m p le x a n d t r a n s f e c t io n .

Vector: Lipofectin- peptide 1 - luciferase plasmid (optimal weight ratio).

Transfection time: 6h Expression time: 42h

Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells.

Incubation of the complex for 30 minutes before it was added to the cells was found to give the highest luciferase activity of the time points which were assessed.

Chapter 4. Optimization of vector system

4.3.3.4. Optimal temperature for formation of transfection complex.

Changing the temperature of complex formation was investigated (Figure 4.15). 4 0 0 0 0 - c B 3 0 0 0 0 - o l- Q. 1* 20000^ Ù1 100001 Control 4 20 37 50 60 Temperature of pre-incubation of complex (°C) F ig u r e 4 .1 5 E f f e c t o f t e m p e r a t u r e o f fo r m a tio n o f c o m p le x e s o n t r a n s f e c t io n .

Vector; Lipofectin- peptide 1 - luciferase plasmid (optimal weight ratio).

Transfection time: 6h Expression time: 39h

Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells.

Room temperature was subsequently used since it was convenient and gave a high activity.

Chapter 4._________________________________________________ Optimization of vector system