Generation of an estrogen receptor beta‐iCre knock‐in mouse

Abstract

A novel knock-in mouse that expresses codon-improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatio-temporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the endogenous start codon of the Esr2 gene of a C57BL/6J embryonic stem cell line via homologous recombination. Resulting Esr2-iCre mice were bred with ROSA26-lacZ and Ai9-RFP reporter mice to visualize cells of functional iCre expression. Strong expression was observed in the ovary, the pituitary, the interstitium of the testes, the head and tail but not body of the epididymis, skeletal muscle, the coagulation gland (anterior prostate), the lung, and the preputial gland. Additional diffuse or patchy expression was observed in the cerebrum, the hypothalamus, the heart, the adrenal gland, the colon, the bladder, and the pads of the paws. Overall, Esr2-iCre mice will serve as a novel line for conditionally ablating genes in Esr2-expressing tissues, identifying novel Esr2-expressing cells, and differentiating the functions of ESR2 and ESR1.

Stimulation of Anxiety-Like Behavior via ERK Pathway by Competitive Serotonin Receptors 2A and 1A in Post-Traumatic Stress Disordered Mice

“…The following day, after washing with TBST, the membrane was incubated with secondary antibody for 2 hours at room temperature (HRP-labeled goat anti-rabbit IgG, HRP-labeled Donkey anti-goat rabbit IgG, HRP-labeled rabbit anti-mouse IgG, Boster Biological Technology Ltd., 1: 8000). Afterwards, protein bands were visualized using an enhanced chemiluminiscence detection kits (ECL-plus, Beyotime Biotechnology, China) by Gel Image Analysis System (Tanon 2500R, Shanghai, China)[17, 37, 38]. …”

Generation and characterization of an estrogen receptor alpha‐iCre knock‐in mouse

“…As RFP expression was observed in various tissues in the adults but only in a few specific cell types at younger ages, the Esr1 –iCre mice may not be used as a deleter line or a cell‐lineage tracer in adulthood but at embryonic or postnatal stages of development. Of note, this novel Esr1 –iCre mouse line may be used along with the Esr 2‐iCre mouse that we previously generated (Cacioppo et al, ) for a lineage‐tracing ESR1‐ vs. ESR2‐expressing cells and for a selective deletion of genes in respective cell lineages.…”

“…An iCre‐polyA‐FRT‐neo‐FRT cassette (3,266 bp) was generated from a pBluescript KS(+)‐iCre plasmid as described in a previous transgenic study (Cacioppo et al, ) and was modified for insertion into the mouse Esr1 gene (Figure a). The cassette was inserted before the translational initiation codon of the Esr1 gene of a BAC clone (ID RP23‐7D5) that was purchased from Invitrogen (Carlsbad, CA) by recombineering according to protocols provided by Frederick National Laboratory for Cancer Research (Jahrling et al, ; Parkitna, Engblom, & Schutz, ; Thomason, Sawitzke, Li, Costantino, & Court, ).…”

Principles of Genetic Engineering

“…Other site-specific recombinases, such as FLP, Dre, and Vika, that work on the same principle have also been applied to mouse models [75][76][77][78][79][80]. Recombinase knockins can be designed to knock out the endogenous gene or preserve its function [81,82]. A variation in the conditional allele is the inducible allele, which is silent until its expression is activated by Cre recombinase [79].…”