Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

Abstract

Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel's Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj's findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis.

Fig 1. Generation of TSPO KO mice.

(A) Schematic of generation of TSPO Floxed mice and conditional KO mice by Cre-LoxP System. Conditional TSPO KO mice were generated by targeting exon 2 and exon 3 of the mouse genomic locus, a NEO selection cassette Floxed by Frt sites. (B) Schematic of crossing program for TSPO global knockout mice, Protamine-Cre is a transgenic mouse where Cre recombinase expression is driven by sperm specific protamine promotor. (C) Genotyping of TSPO Floxed mice and KO mice by a TSPO genotyping primer pair 1 & 3, and Flp primers. (D) Genotyping of TSPO Floxed mice and KO mice by three TSPO genotyping primers 1, 2 and 3, and Protamine-Cre primers.

Fig 2. TSPO deletion does not affect viability by Het x Het and KO x KO breeding.

(A) Birth rate of offspring of TSPO Het x Het breeding. (B) One litter Pups born 4 days from TSPO KO x KO breeding. (C) One litter pups born 9 days from TSPO KO x KO breeding. (D) Birth rate of offspring in TSPO KO x KO breeding. Chi-Squared Test was used to determine significant difference compared with Mendel’s Law.

Fig 3. TSPO expression was abolished in global KO mice without pathological changes.

TSPO expression in different tissues from WT and KO mice were detected by western blotting (A) and IHC (B); (C) H&E staining of different tissues from WT and TSPO KO mice. Scale Bars, 100μm.

Fig 4. TSPO KO did not affect gene expression profiles.

(A) TSPO gene locus of WT and KO mice lung tissue samples used for RNA-seq, results visualized by Integrative Genomics Viewer (IGV) software (NIH), C: coverage of reads, J: junctions, H: hits; (B) Expression level of potential TSPO interaction proteins reported by literatures and SRING database (Version 10.0); (C) Expression profile of bronchoalveolar immune microenvironment associated genes; (B&C) Significant difference determined by negative binomial distribution test.

Fig 5. TSPO KO mice show normal alveolar macrophage population.

(A) FACS analysis of BALF and alveolar macrophages was identified as F4/80+, CD206+. (B) The percentages of alveolar macrophages in BALF from WT and TSPO KO mice are shown as the mean ± S.E.M. from four animals in each group (n = 4). NS, not significant by Student’s t-test.