Establishment Of A 5-lipoxygenase Transgenic Mouse Model

ObjectiveAtherosclerosis (Atherosclerosis, AS) is a human-health-threatening disease and a potential factor which may induce diseases such as myocardial infarction, stroke and etc. so far, the research focus at home and abroad concentrates primarily on AS formation mechanism from the aspect of gene induction, and the research evidences in recent years show that 5-lipoxygenase (5-LO) pathway play a very important role in the formation and development of atherosclerosis, and instability of atherosclerosis plaque. It was proved that 5-LO gene is closely related to AS pathogenesis by using 5-LO knockout mice experiment, even though the 5-LO-defect mice cannot stimulate a series of inflammatory reactions process of patient s which the state of high 5-LO gene expression caused, the model of the 5-LO over-expression transgenic mice can overcome the weakness of gene-knockout mice, and may become the ideal mode for studying the mechanism of AS inflammation. This research stabilizes transfected RAW264.7 cells by using fusion expression vector pEGFP-5LO and initially study the situation of its expression, and also constructs the mode 5-lipoxygenase Transgenic Mouse using the microinjection which lays a good foundation for exploring the effect of 5-LO gene on atherosclerosis prevention.MethodsFirst, total RNA extracted from human peripheral blood as a template, according to GenBank of 5-LO gene sequence, we design a pair of primers, and amplified a 2000bp full-length of 5-LO gene by RT-PCR, then clone it into pUCm-T vector, the sequence of 5-LO was confirmed by the accuracy. pEGFP-C2 and pUCm-T-5-LO digested with EcoR I, and then recovery, ligation, transformation, identification, finally constructed pEGFP-5-LO expression plasmid successfully; the pEGFP-5-LO expression plasmid stability transfected mouse macrophage cell line RAW264.7 cells the empty vector pEGFP-C2 as control, RT-PCR and Western-blot were performed to detectde 5-LO in the expression of RAW264.7 cells. We use the QIAquick Gel extraction Kit purified pEGFP-5-LO plasmid fragment which digested by StuI about 6.8kp, then impact the foreign gene into fertilized eggs by microinjection, after that the injection of fertilized eggs transplanted to the same period in tubal pregnancy in pseudopregnant female mice, Genomic PCR and Southern blot were performed to analyze integration of the target gene into the transgenic DNA of the host. Finally we analyze 5-LO in transgenic mice organizations characteristics and expression of transcription by RT-PCR, Western-blot, immunohistochemistry.ResultThough enzyme digestion, sequencing analysis recombinant plasmid are in line with expectations. The pEGFP-5-LO expression plasmid was successfully constructed. Stable transfection of 5-LO in the RAW264.7 cell line selected by G418, We detected 5-LO mRNA transcription activity was significantly higher than the control which transfected with empty vector pEGFP-C2 of RAW264.7 cells increased (P<0.05) by RT-PCR, Further with the Western-blot to detect 5-LO protein expression levels,it also significantly higher than its control (P<0.05), the result shows the successful transfection and 5-LO gene are expression.166 eggs were injected by microinjection and 90 fertilized eggs were injected transplanted into three of the oviduct of ICR recipient mice.Out of 25 F0 transgenic mice.7 were genomic PCR and Southern-blot positive for 5-LO:1,9,13,17,20,22,24.Though RT-PCR method we found that transgenic mice bone marrow cells, peritoneal cells, kidneys, spleen 5-LO, FLAP, and related factors downstream mRNA transcription increased compared with normal mice. Though western-blot method we found that transgenic positive mouse bone marrow cells, peritoneal cells, kidneys, spleen tissue of 5-LO, FLAP protein expression was significantly higher than the normal mice (P<0.05); We also found positive transgenic mice in the kidney and spleen tissues of 5-LO, FLAP protein expression was significantly higher than normal mice by immunohistochemistry.ConclusionpFGFP-5-LO transfected RAW264.7 cells stability in vitro, can be effective in 5-LO in R NA and protein levels have high expression than normal RAW264.7 cells; 5-LO are integrated and expressed in the mouse genome,5-LO Pathway related factor to rise,5-LO proteins highly expressed in normal mice, indicating successful establishment of a 5-LO overexpression transgenic mice.