The construction, characterization and transfection of liposome- polycation-CDKN1B plasmid complexes.
OBJECTIVE
To develop an efficient non-viral gene delivery system in order to transfer CDKN1B gene efficiently into lung and liver carcinoma cells.


METHODS
A recombinant plasmid composed of CDKN1B sequence and EYFP as reporter gene was constructed and identified. The recombinant DNA was then formulated the lipids-polycation-DNA complexes(LPDs) with protamine sulfate. Several kinds of lung and liver carcinoma cells were transfected by means of LPDs. The physicochemical properties of LPDs were investigated using PCS method and TEM, respectively. The expression of EYFP in A549 cells was observed under fluorescent microscope and evaluated by flow cytometry analysis. Finally, the production of CDKN1B protein in transfected LLC, Chang and 7721 cells was identified by Western blot analysis.


RESULTS
The average diameter of the LPDs were 167 nm with the polydispersity index of 0.35. The average zeta potential of LPDs was +32.6 mV. LPDs look like a sunken sphere. The fluoresent microscope picture clearly indicated the expression of EYFP in A549 cells. The flow cytometry result showed that the transfection efficiency of LPDs in A549 cells was comparable with that of LipofectAMINE, the positive control. Western blot analysis confirmed the production of CDKN1B protein in LLC, Chang and 7721 cells transfected with LPDs, while no CDKN1B protein was detected in cells transfected with naked DNA.


CONCLUSIONS
The construction of the recombinant plasmid is successful. LPDs can deliver the recombinant plasmid to lung carcinoma cells and liver carcinoma cells with high efficiency. Therefore, this kind of gene delivery system has the potential uses for the treatment of lung and liver cancer.
Zhang
Ling
L
School of Life Sciences, Fudan University, Shanghai, PR China.
Sun
Xun
X
Zhang
Hong-wei
HW
Sang
Ying-ying
YY
Yi
Tao
T
Qiao
Shou-yi
SY
D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011479:Protamines / Q000737:chemistry; D014162:Transfection / Q000379:methods*
D050760:Cyclin-Dependent Kinase Inhibitor p27; D049452:Green Fluorescent Proteins; D008081:Liposomes; D011073:Polyamines; D000071228:Polyelectrolytes; D011479:Protamines