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Abstract
The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del16qB3Δ/16qB3Δ) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del16qB3Δ/16qB3Δ animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del16qB3Δ/16qB3Δ hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions.
Highlights
Chromosomal deletions are a powerful tool to assess the biological functions and genetic interactions among contiguous genomic elements irrespective of whether they are protein-coding or non-coding
We took advantage of a previously engineered mouse embryonic stem cells (ESCs) line containing a chromosomal deletion to assess the in vivo biological functions of Csta and Stfa2l1 as well as Fam162a, Ccdc58, and associated intergenic regions (Figure 1A)
Representative flow cytometry profiles of peripheral blood cells stained with antibodies specific to allelic forms of the cell surface marker CD45: CD45.1+ Pep3b recipient mouse, CD45.2+ C57BL/6J control mouse, and immunophenotyping performed 12 weeks following the transplantation of the indicated ratio of Del16qB3D/16qB3D (CD45.2+) and wild-type (CD45.1+) fetal liver cells
Summary
Chromosomal deletions are a powerful tool to assess the biological functions and genetic interactions among contiguous genomic elements irrespective of whether they are protein-coding or non-coding. Microarray-based expression profiling suggests that Fam162a and Ccdc are expressed during mouse embryogenesis and in a wide variety of tissues of adult animals [4]. Enhanced Fam162a cytoplasmic expression is detected in human gastric carcinoma and adenoma compared to normal mucosa but the role of this protein in tumorigenesis is undetermined [5]. The stefin proteins, a subgroup (Type 1) of the cystatin superfamily, act as cytoplasmic inhibitors of cysteine proteases such as cathepsins [6,7]. Given their inhibitory role toward cathepsins, stefin proteins could regulate antigen presentation processes involved in immune response and autoimmune diseases [7,8]. No mouse models deficient in Fam162a, Ccdc, Csta, or Stfa2l1 have been described
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