FIG 3.

A targeted mutation in the Tpcn1^T159 gene abolishes Tpcn1 expression. (A) Schematic representation of Tpcn1^T159 gene structure based on the gene with ID no. 252972 and the cDNA sequence with GenBank accession number NM_145853.2. Red blocks, knockout cassette (SA, splice acceptor; IRES, internal ribosomal entry site; pA, polyadenylation signal). The neomycin resistance gene (Neo) is under the control of a promoter present downstream of the LacZ gene. Vertical segments, exons; unfilled boxes, UTRs. (B) Genotyping of mice for WT and Tpcn1^T159 alleles. Numbered black blocks, coding exons; blue lines, PCR-amplified regions. (C, D) RT-PCR results for expression of Tpcn1 transcripts in tissues from WT or Tpcn1^T159 homozygote mice. Blank, reactions with no RNA; numbered white boxes, noncoding exons. (C) Expression of the truncated chimeric Tpcn1A-LacZ transcript from the Tpcn1^T159 allele. (D) Expression of the Tpcn1A/B transcript determined by probing for regions downstream from the gene targeted disruption. (E) Copy number of Tpcn1A/B transcripts detected in liver from WT and homozygote Tpcn1^T159 animals using RT-qPCR. Bars correspond to the mean ± SEM for 4 animals. (F) Immunoblotting analysis of TPC1A/B present in membrane samples from tissues of WT or Tpcn1^T159 homozygote mice using an anti-TPC1 antibody recognizing the C-terminal region of TPC1 proteins. Red markings, immunoreactive bands not present in Tpcn1^T159 samples.