Expression Of Fatty Acid Desaturase FAD2 Gene In E.coli And Primary Establishment Of Transgenic Mice

BACKGROUND/AIM OF RESEARCHMammals only can pruduce saturated fatty acids and monounsaturated fatty acids. They cannot naturally produce essential fatty acids - beneficial nutrients found mainly in plant and fish oil, so they must rely on a dietary supply. Here we want to establish transgenic mice carry a tissue-specific expression gossypium hirsutum ω -6 fatty acid desaturase(FAD2) gene which can add a double bond into an monosaturated fatty-acid hydrocarbon chain and convert oleic acid to linoleic. Those mice will be a model to establish a pathway of fatty acids metabolism.A prokaryotic expression vector will be established at the same time. On one hand, antibody is necessary to western blot assay. The protein can be used to generate antibody. On the other hand, ω-6 fatty acid desaturase injection assay will be performed to check whether a metabolic pathway is established.METHORDS OF RESEARCHThree plasmids vector with expression elements are used to establish a eucaryotic expression vector by restriction enzyme cutting and ligation. This vector is used to pronucleus microinjection. Founder mice will be examined by polymerase chain reaction(PCR) and southern blot.The pET30 a+ prokatyotic expression vector with a insertion fragment of FAD2 cDNA coding sequence is constructed to express a fusion FAD2 protein with a N-terminal s-tag. With this tag, the protein is easy to detect and purify.RESULTSAfter successfully construction of the eucaryotic expression vector , fertilized eggs were microinjected with linear expression vector and transferred into recipient mice, 34 mice were born and survival. Results of PCR showed that 3 of 34 were positive. Southern hybridization did not show positive. From May the twentith, we begined another microinjection and embryonic transfer and the nearest newborn mice will be cheked at early July.A prokaryotic expression vector was succsessfully constructed and transformed in to E.coli expression strain BL21(DE3). With the IPTG induction, a 50kd protein of ω-6 fatty acid desaturase is expressed.