Journal: BMC Nephrology
Article Title: A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid
Figure Lengend Snippet: ( A ) Detection of exogenous pCAG/DT-A by PCR analysis. Genomic DNA was extracted from organs of the mice injected with pCAG/DT-A/FuGENE™6 complex or PBS(-) (mock injection). Detection of pCAG/DT-A was performed by PCR using DTA-S/DTA-2RV primer set (see Figure 1A ). This PCR yielded a product of 267 bp [indicated by an arrow in ( A )]. C indicates that tail DNA (1 μg) from a B6C3F1 mouse was directly subjected to PCR as a negative control. PC indicates that pCAG/DT-A (4 ng) was subjected to PCR as a positive control. ( B ) Detection of DT-A mRNA by RT-PCR analysis. Detection of DT-A mRNA was performed by RT-PCR using βA-1/DTA-RV primer set (see Figure 1A ), which yielded an expected band of 300 bp [indicated by an arrow in ( B )]. In the lower panel of ( B ), the expression pattern of endogenous β-actin mRNA is indicated. Lu, lung; Br, brain; H, heart; K, kidney; In, intestine; L, liver. C-1 indicates that tail DNA (1 μg) from a B6C3F1 mouse was directly subjected to PCR. C-2 indicates that tail DNA (1 μg) from a B6C3F1 mouse was directly subjected to reverse transcription and the resulting solution was then PCR-amplified. PC indicates that pCAG/DT-A (4 ng) was subjected to RT-PCR as a positive control. A few non-specifically amplified bands above the 300-bp band were observed in lane C-2, and approximately 600-bp bands [indicated by an arrowhead in ( B )], probably corresponding to the products obtained through amplification of pCAG/DT-A, were also seen in lane PC.
Article Snippet: Forty μl of FuGENE™6 (Boehringer Mannheim GmbH, Mannheim, Germany), a transfection reagent (lipid), was diluted with 60 μl of PBS(-), and then added to 20 μg of circular plasmid DNA dissolved in 100 μl of PBS(-) according to the manufacturer's protocol.
Techniques: Polymerase Chain Reaction, Mouse Assay, Injection, Negative Control, Positive Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification