Female C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were fed a chow diet as described above unless otherwise indicated. For knockdown experiments, we used a method developed by Invitrogen that effectively deliverers stealth small interfering RNA (siRNA) to the liver of live animals via Invivofectamine reagent. The prepared siRNA oligonucleotides (7 mg/kg) were injected into 8-week-old female mice via the retro-orbital sinus. The sequences of siRNA oligonucleotides (Invitrogen) are as follows: for mouse G0S2, sense 5′-CAUGCUGUUUCAAGGUGCCACCGAA-3′ and antisense 5′-UUCGGUGGCACCUUGAAACAGCAUG-3′. Control oligonucleotides with comparable GC content were also obtained from Invitrogen. For overexpression experiments, mice were injected with 2.5 × 109 plaque-forming units of adenovirus encoding murine G0S2 (Ad-G0S2) or a control adenovirus (Ad-null) via the retro-orbital sinus. The animal protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee.
Invivofectamine reagent
Manufactured by Thermo Fisher Scientific
Invivofectamine reagent is a transfection reagent designed for in vivo delivery of nucleic acids, such as plasmid DNA and siRNA, to target tissues in animal models. It is formulated to facilitate efficient uptake and expression of the delivered genetic material.
Automatically generated - may contain errors
2 protocols using invivofectamine reagent
1
Liver-Targeted siRNA and Adenoviral G0S2 Manipulation
2
Evaluating Synovial Fibroblast Engraftment
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Animal experiments followed institutional guidelines and were approved by Animal Care and Use Committee of Hospital 12 de Octubre with protocol reference PROEX 407/15. Air pouches were generated on the back of 8- to 10-week-old NOD scid gamma (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) (NSGTM) mice by subcutaneous injection of 3 ml sterile air on day 0 and were maintained by reinjecting 2 ml of sterile air on day 3. On day 4, RASF suspensions (2 × 106 cells in 0.5 ml PBS) were injected into the air pouch cavity. At day 9, 15 μg of HIF-1α or control scrambled siRNA duplexes combined with Invivofectamine reagent (Invitrogen) in 200 μl of 5% glucose were injected into the air pouch cavity. Mice (n = 9 per group) were sacrificed on day 11 and the air pouch cavity membrane together with the subcutaneous tissues was dissected and snap-frozen in OCT compound (Sakura, Alphen aan den Rijn, Netherlands).
Frozen sections were fixed in 4% paraformaldehyde and analyzed by immunoperoxidase labelling with anti-human nuclei antibody (Millipore, Temecula, CA, USA). Sections were counterstained with haematoxylin. The whole area of each tissue was photographed and digitalized using a Zeiss Axiocam ERc5s camera and Zen 2012 software (Zeiss, Jena, Germany) on a Zeiss Axio Scope.A1 microscope, and the number of human RASF nuclei per air pouch wall area was quantified.
Frozen sections were fixed in 4% paraformaldehyde and analyzed by immunoperoxidase labelling with anti-human nuclei antibody (Millipore, Temecula, CA, USA). Sections were counterstained with haematoxylin. The whole area of each tissue was photographed and digitalized using a Zeiss Axiocam ERc5s camera and Zen 2012 software (Zeiss, Jena, Germany) on a Zeiss Axio Scope.A1 microscope, and the number of human RASF nuclei per air pouch wall area was quantified.
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