CIVIL ENGINEERING 365 ALL ABOUT CIVIL ENGINEERING

[ad_1]

Cell culture and transfection

Cell culture and transfection were performed as previously described10. Briefly, HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) with 10% fetal bovine serum (FBS) (Thermo Scientific), 2 mM l-glutamine and non-essential amino acids (Life Technologies). MOLT-4 cells were cultured in RPMI Medium 1,640 (Life Technologies) with 10% FBS. HEK293T cells were transfected with Lipofectamine when cell confluency was ~ 70%. MOLT-4 cells were transfected with Lipofectamine LTX Plus Reagent (Life Technologies)10.

Construction of expression vectors

Exo-Tat is a recently engineered exosomal protein which a membrane associated peptide was added to the N-terminus of Tat and a nuclear localization signal was added to its C-terminus10. cDNA sequences encoding Tat or Exo-Tat were subcloned into an expression vector pAAV-MCS (System Biosciences) between EcoRI and BglII sites resulting in expression vector pAAV-Tat or pAAV-Exo-Tat. For convenience of detecting Tat or Exo-Tat protein, an HA-tag was added to the C-terminal end of Tat or Exo-Tat. The expression vectors were sequenced at Yale Keck Sequencing Facility.

Production of AAV viruses

AAV-Tat or AAV-Exo-Tat viruses were generated by co-transfection the expression vector pAAV-Tat or pAAV-Exo-Tat with pAAV-DJ and pHelper (System Biosciences) at 1:1:1 ratio into HEK293T cells. All the AAV viruses were produced at the cGMP facility of Vigene Biosciences. The titers of AAV-Tat were 3.9 × 1012 GC/mL and 6.95 × 1012 GC/mL. The titers of AAV-Exo-Tat were 2.17 × 1012 GC/mL and 1.97 × 1012 GC/mL. Each vial of AAV was in 250 μL PBS.

Animals

The experimental protocol was approved by the Lifespan Animal Care and Use Committee. The approval number is 505517. All experimental procedures were conducted in accordance with guidelines for the ethical treatment of animals. Balb/cj mice (3 months old) were purchased from Jackson Laboratory. Vehicle control or AAV viruses were injected into mice via tail veins (n = 3, 1 × 1012 GC/mouse in 200 μl PBS). The mice were kept in a BL2 animal facility at Rhode Island Hospital and taken care by a certified technician. The mice were euthanized on day 21 by overdose isoflurane. Blood was taken by cardiac puncture before death. Brain, heart, intestine, kidney, liver, lung, muscle and spleen were taken after death.

Semiquantitative RT-PCR

Total RNA was extracted from mouse organs with TRIZOL reagent (Invitrogen) and 1 μg of total RNA was used for cDNA synthesis using MMLV reverse transcriptase (New England Biolabs) as described in the manufacturer’s manual. Two μl of cDNA was used for PCR reaction to check Tat or Exo-Tat expression. GAPDH was used as control for normalization.

Western blotting (WB)

WB was performed as previously described21. Briefly, cell or tissue lysates were separated by 10% SDS–PAGE electrophoresis and electroblotted to nitrocellulose membrane (Bio-Rad). Blotted membranes were probed with their respective primary antibodies, rotating at 4 °C overnight. Membranes were washed three times in TBST buffer and probed with secondary antibody (IRDye800-conjugated Affinity Purified IgG) at room temperature for 1 h. Membranes were then washed three times in TBST buffer and direct infrared fluorescence detection was performed with a Licor Odyssey Infrared Imaging System21.

Tat ELISA

Since we couldn’t find a good commercial Tat ELISA kit. We used a modified Tat ELISA protocol. Briefly, 96-well plate (USA Scientific) was coated with 0.2 μg HA-tag rabbit monoclonal antibody (Cell Signaling) per well. Wash the wells with TBST and block the wells with blocking buffer (Licor) for 1 h at room temperature. Add 100 μL sample to specific wells and incubate at 4 °C overnight. After washing with TBST, Anti-HIV1 tat antibody (Biotin) (Abcam) in blocking buffer with 1:100 dilution was added to each well and incubate at room temperature for 2 h. Wash the wells with TBST for three times. Incubate with 100 μl diluted Streptavidin-HRP (1:100 dilution) (PerkinElmer) for 30 min at room temperature. After washing with TBST for 3 times, incubate with 100 μl OPD (O-Phenylenediamine Dihydrochloride) (PerkinElmer) for 30 min at room temperature, add 100 μl stop solution (PerkinElmer) to terminate the reaction, measure the absorbance at wavelength 490 nm using SpectraMax M5.

Statistical analysis

Quantitative data were analyzed by unpaired Student’s t test to compare two groups. Data are expressed as mean ± standard error of mean. A p value < 0.05 indicates statistical significance.

[ad_2]

Source link

Leave a Reply

Your email address will not be published. Required fields are marked *