Journal: Scientific Reports
Article Title: Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors
doi: 10.1038/srep41270
Figure Lengend Snippet: ( A ) Structure of the salivary gland-specific human NGF (hNGF) plasmid pmPSP-hNGF. The transposon is flanked by the pB 5′TRE and pB 3′TRE, the piggyBac transposon 5′ and 3′terminal repeat elements. It was assembled to contain (5′ to 3′): PSP, the mouse parotid secretory protein gene promoter, which is salivary glands specific; the hNGF gene; the bovine growth hormone gene poly-A signal (bGH-pA); the cytomegalovirus promoter (CMV) driving the Neomycin-resistance gene and EGFP gene, linked by a 2 A peptide (Neo-2A-EGFP); and finally a bGH-pA. The location of primer set #1 (P1 + P2), #2 (P3 + P4), and #3 (P5 + P6), which were used for PCR, qPCR/RT-PCR and inverse PCR respectively, as well as the probe and enzyme used for Southern blot are also shown on the plasmid map. ( B ) PCR identification of TG F 0 founder mice. N represents negative control using water as template, P positive control using plasmid pmPSP-hNGF or pm PB as template, M represents molecular markers and Rgs7 is for the regulator of G protein signaling 7, which was used as an internal control gene. ( C ) EGFP expression in the claw tissues of TG F 0 mice. ( D ) Analysis of transgene integration patterns in the genome of TG F 0 mice by Southern blot. M depicts molecular markers. P (3 C) and P (5 C) are samples where three copies (22.3 pg) or five copies (37.2 pg) of the plasmid were added to 10 μg of WT mouse genomic DNA as positive controls. The absence of a positive signal for 563 and 564 TG F 0 mice could be due to degradation of their genomic DNAs as their samples were isolated from the postmortal tail tissues, while all other genomic DNA samples were extracted from live mice’s tail biopsies.
Article Snippet: Detection of hNGF on the membrane was carried out by using monoclonal rat anti-hNGF primary antibody (Cat. #MAB2562, R & D systems, Minneapolis, MN, USA), HRP-conjugated goat anti-Rat secondary antibody (Jackson Immuno Research, West Grove, PA, USA), and SuperSignal West Pico Chemiluminent Substrates (Thermo Scientific Pierce, Guangzhou, China) following the manufacturer’s protocols. mNGF1 (Staidson, Beijing, China) which was isolated from mouse submandibular glands and is currently an approved human drug for sale in China, and mNGF2 (Cat. #1156-NG, R & D systems, Minneapolis, MN, USA) which was expressed and purified from mouse myeloma cells were used as controls.
Techniques: Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Inverse PCR, Southern Blot, Negative Control, Positive Control, Control, Expressing, Isolation
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