Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.

[Establishment of a 293-cell line containing luciferase reporter for EIAV receptor and LTR functions].

To accurately and conveniently detect neutralizing antibodies and receptor binding affinities of different equine infectious anemia virus (EIAV) strains, the cDNA of EIAV receptor, ELR1, was cloned and inserted in an eukaryotic expression vector pcDNA3.1(+). This recombinant plasmid was designated as pELR1. The 293 cell line was transiently transfected with pELR1 and the expression of ELR1 on transfected cells was verified by Western blot and indirect immunofluorescence assay (IFA). Furthermore, the transcription regulatory region, long terminal repeat (LTR), of an EIAV vaccine strain and a reporter of firefly luciferase gene were tandemly cloned into pELR1. The resultant expression vector, which was designated as pELR1-LTR-Luc, was used to transfect 293 cells. A transfected cell line, ELR1-LTR-Luc (293E) which consistently expressed ELR1 and produced luciferase under the regulation of LTR, was isolated and further characterized. The entrance and replication of EIAV in ELR1-LTR-Luc (293E) cells were verified by IFA. The luciferase activity in the cell line treated with 1000 TCID50 of an EIAV vaccine strain for 24h was increased by 2.15 folds when compared with the activity in untreated cells. Furthermore, the luciferase activities in the cell line were linearly correlated with the doses of inoculated EIAV virulent stain L21 diluted at 10(-2) to approximately 10(-7). The transfected genes in the ELR1-LTR-Luc (293E) cell line were consistently expressed during 35 passages of the host cells. This ELR1-LTR-Luc report system can be used for the study of interaction between EIAV strains and the receptor, as well as for the evaluation of neutralizing antibodies raised by EIAV.

[Construction and rescue of rabies virus mutant strain SRV9].

To construct a rabies virus mutant, the psi region was replaced by the coding region of human cytochrome c gene, and the coding region for cytoplasmic domain of glycoprotein G was deleted in the full-length of genomic cDNA of rabies virus strain SRV9. The mutant plasmid and the plasmids with N, P, L and G structural proteins of wild type SRV9 were co-transfected into BHK-21 cells. It was shown by IFA that there were many specific fluorescence in the BHK-21 cells, and typical rabies virus virions were observed by electronic microscope. These results demonstrated that the mutant rabies virus was successfully rescued. The genetically modified SRV9 stain has promise to provide invaluable experimental tool to develop attenuated live rabies vaccine.