Abstract:

BACKGROUND: Studies have shown that decreased expression of ribosome protein S14 (RPS14) gene in hematopoietic stem/progenitor cells can lead to the dysplasia of hematopoietic cells, in particular the blocked erythroid differentiation. Normal expression of RPS14 gene can help to maintain the normal hematopoiesis.
OBJECTIVE: To construct a RNA interference lentiviral vector of RPS14 gene, and to observe its expression in the human myelodysplastic syndrome cell line SKM-1 cells.
METHODS: The targeting sequence of RPS14 gene which effectively silenced in RNA inference was confirmed in our previous study. The GC-shRPS14 lentiviral vector was established and sequenced. The 293T cells were co-transfected with lentiviral vector GC-shRPS14, pHelper 1.0 and pHelper 2.0. The titer of virus was tested according to the expression level of GFP. The resulting S14shRNA lentiviral vector GC-shRPS14 was used to infect human myelodysplastic syndrome cell line SKM-1. And the RPS14 gene expression in the target cells was detected by Western Blot.
RESULTS AND CONCLUSION: DAN sequencing demonstrated that the lentiviral vector GC-shRPS14 of RPS14 shRNA was constructed. The titer of concentrated virus was 2×109 Tu/ml. GFP expression could be seen in the transfected SKM-1 cells under fluorescence microscope, and the transfection efficiency was 75% Western Blot showed that. RPS14 expression was significantly inhibited in the target cells after transfection. The RNA interference lentiviral vector of RPS14 was constructed successfully, which is capable of delivering the target gene RPS14 into human myelodysplastic syndrome cell line SKM-1 for its silent expression.