| ObjectivesThe plasmid pBSK5mOVA was transfected into the murine skin tissues in vivo and expressed in murine skin tissues. This work will found the basis of the next making animal disease model induced by the protein encoded by this plasmid.Materials and Methods1. Plasmid DNA: the plasmids pBSII and pBSK5mOVA , the plasmid pBSII was a carrier plasmid containing the Escherichia coli( E. Coli ) -galacto-sidase gene ( LacZ gene) and ampicillin resistance gene ( AMPr) . The plasmid pBSK5mOVA was a recombinant plasmid containing the human K5 promoter, OVA peptide gene and human growth factor' s poly-A.2. Animals and bacterian strain; inbred lines C57BL/6 and BALB/c, 6-8 weeks old, weight 15-20 g, Male, they were bought from the Animal Lab of China Medical University. The strain was DH5ct strain of the E. Coli.3. Main reagents and RT-PCR primer: TRIZOL ( Invitrogen company, USA) , Access RT-PCR System kit ( Promega company, USA ) , lipofectamin 2000 ( Invitrogen company, USA) , Rabbit anti-chicken egg ovalbumin poly-clonal antibody ( Life sciences company, USA) , RT-PCR primer was designed according to the literature.4. Amplification , extraction and purification of plasmids: firstly, DHSct strain of the E. Coli was prepared into competent cells(CCs) by CaCl2method; Secondly,the plasmid pBSII and pBSK5mOV were transformed into the CCs,the CCs grew and breeded in Luria-Bertani ( LB) culture medium; Screening the plasmids by blue-white colonies, blue colonies contains the plasmid pBSII,white colonies contains the plasmid pBSK5mOVA. DNA was extracted from the colonies by base lyses method and electrophoresed on 0. 8% agarose gel to analyze the length of plasmid DNA. The longer length of plasmid was cut with restriction endonuclease BamHI; the cutting reaction solution was electrophoresed on 1% agarose gel to analyze the length of enzyme-cutting DNA fragments.5. The plasmid pBSII transfection in vivo and detection: Experimental animals were BALB/c mice and randomly divided into threegroups: (1) experiment group: the belly was depilated and applied topically with the plasmid pBSII-lipofectamin鈩?000 formulation, twice one day for 3 days. (2) control group: the belly was depilated and applied topically with the naked plasmid pBSII, twice one day for 3 days. (3) negative group: the belly was only depilated . On the 4th day, the belly skin tissues were harvested, fixed, and stained in X-gal solution, then the tissues were processed into paraffin sections and observed whether there were or not blue staining cells under light microscope.6. The plasmid pBSKSmOVA transfection in vivo and detection: Following successful transfection of the short plasmid pBSII, we transfectedthe plasmid pBSK5mOVA into C57BL/6 and BALB/c mice with lipofectamin鈩?2000. There were 5 groups; (1) C57BL/6 mice, the belly was depilated and topically applied the lipofectamin鈩?000. (2) C57BL/6 mice, the belly was depilated and topically applied the plasmid pBSK5mOVA. (3) C57BL/6 mice, the belly was depilated and topically applied the plasmid pBSK5mOVA- lipo-fectamin鈩?000 formulation. (4) C57BL/6 mice, the belly was shaved the hair and topically applied the plasmid pBSK5mOVA- lipofectamin 2000 formulation. (5) BALB/c mice, the belly was depilated and topically applied the plasmid pBSK5mOVA- lipofectamin鈩?000 formulation. Every group mice were applied twice one day for 3 days. On the 4th day, the applied skin tissues were shaved and divided into 2 pieces, one for total RNA extraction with TRIZOL reagent, the total RNA was measured content and purity by detecting the optical density at 260nm and 280nm, then the total RNA was reverse transcripted into cDNA and amplified by PCR. RT-PCR products were electrophoresed on 2% agarose gel; Another one was prepared into freezen-sections for immunohisto-chemical Staining, we choose AEC to show color and applied ABC peroxidase technique with rabbit anti-chicken egg ovalbumin polyclonal antibody for ovalbu-min expression.Results1. Amplificati...
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