PMC:2669177 / 9181-11791 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/2669177","sourcedb":"PMC","sourceid":"2669177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2669177","text":"We next examined the identity of the cells that took up the exogenously delivered NMP cDNA and the efficiency of gene product expression within the retina over time, using immunohistochemistry. The entire eye was cut and every sixth section was collected and assessed, enabling us to examine gene expression in multiple regions throughout the retina. Due to an epitopic modification in the NMP carboxyl terminus (P341Q), which is not present in wildtype RDS and does not result in retinal disease or vision loss [24], the transferred Rds gene product can be detected selectively even on a normal RDS background using a monoclonal antibody (mAB 3B6) that recognizes the P341Q epitope. [For demonstration of the selectivity of mAB 3B6 for transferred RDS (NMP) as opposed to native (endogenous RDS), see Figure S2 and our previous publications [24], [34].] The endogenous mouse RDS protein (and to a much lesser extent transgenic NMP protein) is labeled with the RDS-CT antibody [35]. Although normal OS development has not yet begun at P7 (PI-2) [36], Figure 2 (top row) shows expression of both transferred (Figure 2A and 2B) and native RDS (Figure 2C) protein in the tip of the photoreceptors. By PI-7 (P12), distinct outer and inner nuclear layers are apparent and NMP/RDS staining in the tips of nascent OSs is visible as a thin immunopositive layer adjacent to the photoreceptor nuclei. NMP distribution in the OSs persisted through the latest time point examined (PI-30). NMP also co-localized with native RDS and was usually limited to the OS layer (Figure 2); no NMP was detected in eyes injected with saline (Figure 2C and Figure S3) or naked DNA (not shown). Occasionally, NMP expression was detected in RPE cells after nanoparticle injection (Figure 2). Expression in the RPE was highly variable; while many animals had some RPE expression, others did not. RPE expression was much more common in eyes injected with CBA-NMP nanoparticles, consistent with its role as a ubiquitous promoter. On the other hand, IRBP-NMP staining in the RPE was almost always limited to PI-2. NMP detection with mAB 3B6 in the OSs was heterogeneous, with stronger signal in areas closer to the injection sites. However, we estimate that a majority of photoreceptors expressed the product of the transferred gene (based on a qualitative assessment of mAB 3B6 immunostaining in successive retinal sections). The choice of promoter did not have any apparent effect on cellular distribution within the photoreceptor: both CBA-NMP and IRBP-NMP nanoparticles exhibited similar distribution patterns at all time points examined.","tracks":[{"project":"2_test","denotations":[{"id":"19390689-15277471-97510220","span":{"begin":513,"end":515},"obj":"15277471"},{"id":"19390689-15277471-97510220","span":{"begin":513,"end":515},"obj":"15277471"},{"id":"19390689-15277471-97510221","span":{"begin":843,"end":845},"obj":"15277471"},{"id":"19390689-15277471-97510221","span":{"begin":843,"end":845},"obj":"15277471"},{"id":"19390689-15656787-97510222","span":{"begin":849,"end":851},"obj":"15656787"},{"id":"19390689-15656787-97510222","span":{"begin":849,"end":851},"obj":"15656787"},{"id":"19390689-15254014-97510223","span":{"begin":978,"end":980},"obj":"15254014"},{"id":"19390689-15254014-97510223","span":{"begin":978,"end":980},"obj":"15254014"},{"id":"19390689-8570600-97510224","span":{"begin":1046,"end":1048},"obj":"8570600"},{"id":"19390689-8570600-97510224","span":{"begin":1046,"end":1048},"obj":"8570600"},{"id":"19390689-15277471-97510220","span":{"begin":513,"end":515},"obj":"15277471"},{"id":"19390689-15277471-97510220","span":{"begin":513,"end":515},"obj":"15277471"},{"id":"19390689-15277471-97510221","span":{"begin":843,"end":845},"obj":"15277471"},{"id":"19390689-15277471-97510221","span":{"begin":843,"end":845},"obj":"15277471"},{"id":"19390689-15656787-97510222","span":{"begin":849,"end":851},"obj":"15656787"},{"id":"19390689-15656787-97510222","span":{"begin":849,"end":851},"obj":"15656787"},{"id":"19390689-15254014-97510223","span":{"begin":978,"end":980},"obj":"15254014"},{"id":"19390689-15254014-97510223","span":{"begin":978,"end":980},"obj":"15254014"},{"id":"19390689-8570600-97510224","span":{"begin":1046,"end":1048},"obj":"8570600"},{"id":"19390689-8570600-97510224","span":{"begin":1046,"end":1048},"obj":"8570600"}],"attributes":[{"subj":"19390689-15277471-97510220","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510220","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510221","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510221","pred":"source","obj":"2_test"},{"subj":"19390689-15656787-97510222","pred":"source","obj":"2_test"},{"subj":"19390689-15656787-97510222","pred":"source","obj":"2_test"},{"subj":"19390689-15254014-97510223","pred":"source","obj":"2_test"},{"subj":"19390689-15254014-97510223","pred":"source","obj":"2_test"},{"subj":"19390689-8570600-97510224","pred":"source","obj":"2_test"},{"subj":"19390689-8570600-97510224","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510220","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510220","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510221","pred":"source","obj":"2_test"},{"subj":"19390689-15277471-97510221","pred":"source","obj":"2_test"},{"subj":"19390689-15656787-97510222","pred":"source","obj":"2_test"},{"subj":"19390689-15656787-97510222","pred":"source","obj":"2_test"},{"subj":"19390689-15254014-97510223","pred":"source","obj":"2_test"},{"subj":"19390689-15254014-97510223","pred":"source","obj":"2_test"},{"subj":"19390689-8570600-97510224","pred":"source","obj":"2_test"},{"subj":"19390689-8570600-97510224","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#a093ec","default":true}]}]}}