[Expression of FasL gene in porcine chondrocytes].
OBJECTIVE
To express the Fas ligand(FasL) gene in porcine chondrocytes.


METHODS
The porcine FasL gene fragment was amplified by RT-PCR and then inserted into the pGCEN retroviral vector. The recombinant vector was transfected into packaging cells PA317 which were then screened with G418. Supernatant of screened PA317 cells containing high titer recombinant virus was used to infect porcine chondrocytes. Expression of FasL in chondrocytes was analyzed by FACS and Western blot.


RESULTS
The recombinant pGCEN-FasL retroviral expression vector was successfully constructed as shown by restriction enzyme digestion analysis and DNA sequencing. FasL was expressed in 57% of infected chondrocytes. Western blot analysis also confirmed the expression of FasL in chondrocytes. The expressed FasL could induce apoptosis of Fas+ cells.


CONCLUSIONS
The recombinant pGCEN-FasL retroviral vector has been successfully constructed and FasL with biological activity was highly expressed in porcine chondrocytes, which lays the foundation for allogenic chondrocytes transplantation.
Wang
Shu-jun
SJ
Shanghai Second Medical University, Shanghai Institute of Immunology, Shanghai 200025, China.
Wang
Ying
Y
Zhang
Hui-zhen
HZ
Jin
Shu
S
Shen
Tian-wei
TW
Ge
Yu
Y
Ge
Hai-liang
HL
D000818:Animals; D019902:Chondrocytes / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D053222:Fas Ligand Protein / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013552:Swine / Q000235:genetics*
D004274:DNA, Recombinant; D053222:Fas Ligand Protein; D004262:DNA Restriction Enzymes