Figure 3. Generation and characterization of the proNgf-HA/+ and wtNgf-HA/+ knock-in mice. (A) Schematic of ngf gene targeting with the prongf-HA allele. The dark bars indicate Southern blot probe sequence and expected product sizes. Inset, Southern blot analysis of genomic DNA. (B) Schematic of wild-type ngf-HA gene targeting. Inset, Southern blot analysis of genomic DNA. (C) RT-PCR analysis to detect proNGF-HA mRNA from embryonic day 16.5, postnatal day 0, and postnatal day 1 mouse hearts. Control, pcDNA vector. proNGF-HA, pcDNA-proNGF-HA plasmid positive control. The experiments were repeated two independent times. (D) Immunoprecipitation–Western blot analysis of proNGF or NGF expression. Left, representative lysates of P0 Ngf+/+ and proNgf-HA/+ hearts (three hearts pooled for each lane) analyzed for proNGF-HA (∼32 kD) and mature NGF-HA (∼13 kD) in proNgf-HA/+ and Ngf+/+ hearts, experiment performed two independent times. Right, similar analysis performed in adult brain lysates. Experiment was performed four independent times. The black line indicates that intervening lanes were spliced out. (E) ELISA for total levels of NGF protein isoforms in the brain and in the heart of Ngf+/+ versus proNgf-HA/+ mice (n = 3 mice/group, mean ± SD).
Figure 3.

Generation and characterization of the proNgf-HA/+ and wtNgf-HA/+ knock-in mice. (A) Schematic of ngf gene targeting with the prongf-HA allele. The dark bars indicate Southern blot probe sequence and expected product sizes. Inset, Southern blot analysis of genomic DNA. (B) Schematic of wild-type ngf-HA gene targeting. Inset, Southern blot analysis of genomic DNA. (C) RT-PCR analysis to detect proNGF-HA mRNA from embryonic day 16.5, postnatal day 0, and postnatal day 1 mouse hearts. Control, pcDNA vector. proNGF-HA, pcDNA-proNGF-HA plasmid positive control. The experiments were repeated two independent times. (D) Immunoprecipitation–Western blot analysis of proNGF or NGF expression. Left, representative lysates of P0 Ngf+/+ and proNgf-HA/+ hearts (three hearts pooled for each lane) analyzed for proNGF-HA (∼32 kD) and mature NGF-HA (∼13 kD) in proNgf-HA/+ and Ngf+/+ hearts, experiment performed two independent times. Right, similar analysis performed in adult brain lysates. Experiment was performed four independent times. The black line indicates that intervening lanes were spliced out. (E) ELISA for total levels of NGF protein isoforms in the brain and in the heart of Ngf+/+ versus proNgf-HA/+ mice (n = 3 mice/group, mean ± SD).

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