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. 2016 Jan;54(1):38-52.
doi: 10.1002/dvg.22911. Epub 2016 Jan 8.

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

Joseph A Cacioppo  1 , Yongbum Koo  1   2 , Po-Ching Patrick Lin  1 , Sarah A Osmulski  1 , Chunjoo D Ko  1 , CheMyong Ko  1
Affiliations

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

Joseph A Cacioppo et al. Genesis. 2016 Jan.

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 spatiotemporal 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.

Keywords: Cre recombinase; Esr2-iCre; estrogen receptor beta; granulosa cells; knock-in.

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Figures

Figure 1
Figure 1. Generation of Esr2-iCre mice
To create mice that faithfully express codon-improved Cre recombinase under regulation of estrogen receptor beta, an iCre-polyA-FRT-PGK-neomycin-FRT cassette was inserted the Esr2 gene. A. First, a targeting vector was generated in a BAC clone by inserting the construct containing iCre into exon 2 [E2] of Esr2. Next the targeting vector was inserted into the genome of C57BL/6J embryonic stem cells using homologous recombination; stem cells were screened for correct insertion location and direction by long range PCR and copy number. Horizontal arrows represent LR-PCR primer binding sites. One clone was then used for blastocyst injection to create a knock-in mouse line. One chimeric mouse that gave birth to multiple black iCre-positive offspring was chosen as a founder and one offspring was crossed with a FLP recombinase-expressing mouse to remove the FRT-PGK-neo-FRT targeting vector. B. The iCre-polyA-FRT-PGK-neomycin-FRT cassette was inserted into exon 2 of the Esr2 gene in a BAC clone and retrieved in a pL253 targeting vector. Homology arms allowing for recombination were added to each end of the cassette by restriction enzyme digestion and end joining. The targeting vector was linearized by Cla1 digestion and inserted into the genome of C57BL/6J embryonic stem cells. C. After homologous recombination, long-range PCR was used to identify ES cell clones that had correct homologous recombination in the 5’-side homology arm of the targeting vector using primers LR-F1 and LR-vR. Positive PCR bands are indicated by the grey arrow in C). Among them, six clones had correct homologous recombination in the 3’-side homology as well (white arrow, primers LR-vF and LR-R1). These six clones were further screened to determine whether the iCre sequence was additionally inserted somewhere else in the genome. Two clones (asterisks corresponding to arrows in D) had one cassette insertion at the correct location and orientation. D. Selection cassette copy number analysis using the neomycin positive marker was used to determine the number of copies of cassette present in each sample and eliminate those with insertions into the genome in addition to the correct location. Two of the clones (black arrows) had a single correct cassette insertion and EsrCre_030 was ultimately used for blastocyst injection.
Figure 2
Figure 2. Expression of Esr2-iCre in the ovary, oviduct, and uterus
Female reproductive organs from post-natal day 60 female Esr2-iCre Ai9 mice were collected; mice were unstimulated and had not previously been pregnant. A,B Fluorescence was grossly observed in the ovary, oviduct, and uterus. C-F Strongest fluorescence was seen throughout the ovary, in the granulosa cells as well as the lutein cells of the corpora lutea and the stromal cells. A few oocytes (~5%) within mature follicles are RFP positive. Arrowhead: antral follicle; asterisk: corpus luteum. G-J Punctate fluorescence was seen in the oviduct, particularly in the proximal oviduct (ampulla). Fluorescence was limited to individual epithelial cells. Arrow: oviduct; arrowhead: ovary; asterisk: adipose tissue. K-N In the uterus, diffuse fluorescence was grossly observed throughout. Expression was observed in luminal epithelial cells lining the endometrium and uterine glands (frozen section). Some mice (>60 days) also displayed fluorescence in the several individual endometrial stromal cells and myometrial smooth muscle cells. Arrowhead: luminal epithelium; asterisk: myometrium; arrow: glandular epithelium. Magnification: E,F: 10×; I,J: 20×; M,N: 10×.
Figure 3
Figure 3. Functional expression of Esr2-iCre in the male gonad and accessory organs
Mature post-natal day 60 male Esr2i-iCre Ai9 mice were sacrificed and organs were collected for histology. A. In the testis, fluorescence was grossly visible outlining the individual seminiferous tubules but not vessels or adipose tissue. Histologically, fluorescence was observed in interstitial cells including Leydig cells but not Sertoli cells or any germ cells (10×). B. The head and tail (caput and cauda) of the epididymis but not the body (corpus) were RFP-positive. Cells of the epididymal epithelium stained in an individual punctate pattern with no fluorescence observed in the smooth muscle cells or the spermatozoa (cauda epididymis, 20×). Arrow: caput; asterisk: corpus; arrowhead: cauda. C. There was weak and scattered staining grossly throughout the ductus deferens. Histologically, fluorescence was limited to a punctate or stippled pattern in only the epithelial cells, similar to the epididymis. Arrow: epithelium; asterisk: spermatozoa; arrowhead: smooth muscle; 20×. D. Grossly after opening the peritoneal cavity, obvious fluorescence was observed in the testis, prepucial gland, caput and cauda epididymis, and was brightest in the anterior prostate/coagulation gland, with weaker fluorescence in the ductus deferens and bladder. No fluorescence was observed in the seminal vesicles, the ureters, or the body of the epididymis. The epithelial cells of the coagulation gland were RFP-positive on histological examination (10×). Solid arrow: coagulation gland; open arrow: seminal vesicle: solid arrowhead: testis; open arrowhead: prepucial gland (cut); solid chevron: caput epididymis; open chevron: corpus epididymis; asterisk: bladder.
Figure 4
Figure 4. Expression of Esr2-iCre throughout the body
Esr2-iCre Ai9 mouse tissues were collected at 2-4 months of age. Expression was similar in the organs of mice of each gender, including the brain and pituitary. Consistent faint fluorescence was observed throughout the detrusor muscle of the bladder. In the brain, neurons of the cerebrum were RFP-positive though this was sharply limited to the forebrain. Ventrally, expression was present throughout the hypothalamus (asterisk) and the ventral portion of the cerebrum; expression was absent from the optic nerve chiasm. Uniquely, expression was observed in bilaterally symmetrical ventral point structures about the olfactory tubercle (solid arrows, ventral brain view). The mucosa of the distal GI tract (colon, cecum), the central interior cornea of the eyes and the nearby conjunctiva (chevron), the epicardium surrounding the coronary arteries and atria of the heart (arrowheads), the entirety of the lungs, select acini of the pancreas, and throughout the skeletal muscle of the body (skeletal muscle in center surrounded by adipose tissue and skin).
Figure 5
Figure 5. Esr2-iCre homozygous mice globally lack Esr2
Esr2-iCre mice were bred together to generate mice homozygous for the iCre allele. Female mice were sacrificed at 26 days old and ovaries were collected. A. The primer locations and band size for Esr2 forward and reverse primers and iCre forward and reverse primers are shown for WT and Esr2-iCre mice. B. Semi-quantitative PCR revealed no Esr2 expression in homozygous iCre mice, and there was no difference in Esr2 expression in heterozygous Esr2-iCre and WT mice. Similarly, iCre expression was not different between heterozygous and homozygous Esr2-iCre mice and was absent in WT mice. Expression of iCre mimics Esr2. Rpl19 was used as an internal control. C. Quantitative expression was determined by semi-quantitative PCR based on band intensity; n=4/group, error bars represent the S.E.M.
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