Generation of prolactin-inducible protein (Pip) knockout mice by CRISPR/Cas9-mediated gene engineering.

Lucas E L Terceiro,Etienne Leygue,Anne A A Blanchard,Chidalu A Edechi,Barbara Triggs-Raine,Agnes Freznosa,Yvonne Myal

doi:10.1139/cjpp-2021-0306

Abstract

Prolactin-inducible protein (PIP) is a multifunctional glycoprotein that is highly expressed and found in the secretions of apocrine glands such as salivary, lacrimal, and sweat glands including the mammary glands. PIP has been implicated in various diseases, including breast cancer, gross cystic disease of the breast, keratoconus of the eye, and the autoimmune Sjögren's syndrome. Here we have generated a Pip knockout (KO) mouse using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRSPR-associated (Cas)9 system. The Cas9 protein and two single guide RNAs targeting specific regions for both exons 1 and 2 of the Pip gene were microinjected into mouse embryos. The deletions and insertions promoted by CRISPR/Cas9 system on the Pip gene successfully disrupted Pip protein coding, as confirmed by PCR genotyping, sequencing, and ultimately Western blot analysis. This mouse model was generated in the inbred C57Bl/6J mouse, which exhibits lower genetic variation. This novel CRISPR Pip KO mouse model will not only be useful for future studies to interrogate the multifunctional role of PIP in physiological processes but will facilitate a broader understanding of the function of PIP in vivo while providing unprecedented insight into its role in a spectrum of diseases attributed to the deregulation of the PIP gene.

Highlights

The prolactin-inducible protein (PIP), known as gross cystic disease fluid protein (GCDFP-15) is a 17 kDa glycoprotein first identified as a secreted protein in some human breast cancer (BC) cell lines (Shiu & Iwasiow, 1985) and in gross cystic disease of the breast (Mazoujian et al, 1983)
We report the generation of a homozygous Pip KO mouse by CRISPR/Cas9 gene editing in the inbred C57Bl/6J mouse strain
The mice were housed in the Central Animal Care (CAC) facility at the University of Manitoba, where they were maintained in a temperature-controlled room with normal light-dark cycles and fed ad-libitum

Summary

Introduction

The prolactin-inducible protein (PIP), known as gross cystic disease fluid protein (GCDFP-15) is a 17 kDa glycoprotein first identified as a secreted protein in some human breast cancer (BC) cell lines (Shiu & Iwasiow, 1985) and in gross cystic disease of the breast (Mazoujian et al, 1983). PIP is encoded by the PIP gene located at the long arm of human chromosome 7q32-36 (Myal et al, 1991) and its expression is regulated by distinct hormones, in particular prolactin, androgen and interleukin (for review, Hassan et al, 2009). The PIP nucleotide sequence and tissue-specific expression were found to be conserved across a diverse range of species including rats and mice (Myal et al, 1991), suggesting that the evolutionary retention of this gene is of biological and physiological importance. PIP is strategically found in tissues considered the first line of defense against invading organisms including the skin, eyes, and mouth which are major routes of pathogen entry in the body (for review, Hassan et al, 2009; Ihedioha et al, 2016).

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