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. 2014 May;155(5):1991-9.
doi: 10.1210/en.2013-2150. Epub 2014 Feb 7.

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing

M A Karim Rumi  1 , Pramod Dhakal, Kaiyu Kubota, Damayanti Chakraborty, Tianhua Lei, Melissa A Larson, Michael W Wolfe, Katherine F Roby, Jay L Vivian, Michael J Soares
Affiliations

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing

M A Karim Rumi et al. Endocrinology. 2014 May.

Abstract

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

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Figures

Figure 1.
Figure 1.
ZFN generated targeted mutations at the Esr1 locus. A and B, Schematic representations of the ZFN target site within the rat Esr1 (NC_005100.3) locus (A) and PCR primer locations (B) used for mutation detection. C, PCR products from founder rat DNA samples were identified by agarose gel electrophoresis and ethidium bromide staining and mutations (mutant animal numbers are in bold) were resolved by DNA sequence analysis. D, Offspring of the monoallelic mutant founder (F871) possessed a wild-type allele and an allele containing either a 482-bp (**) or a 223-bp (*) deletion at the Esr1 locus, indicating mosaicism. E and F, DNA sequence analysis showing the 482-bp deletion (Δ482) (E) and the 223-bp deletion (Δ223) (F) at the Esr1 gene locus. Schematic diagrams beneath each DNA sequencing profile show the impact of the deletion on exon 3 of the Esr1 gene. E, The nucleotides in blue reflect the 9-bp remnant of exon 3 for Δ482. F, In contrast, Δ223 contains an exon 3 that is largely intact except for a 9-bp truncation at the 3′ end of the exon. The terminal 9 nucleotides of remaining exon 3 for the Δ223 deletion are highlighted in blue.
Figure 2.
Figure 2.
ESR1 deficiency in rats possessing homozygous Esr1 mutations. A, Schematic representation of the Esr1 gene (NC_005100.3) and the locations of primers used for RT-PCR. B and C, RT-PCR analyses for Esr1 detected a weakly expressed shorter transcript in uterine tissues from Δ482 mutants (B), whereas multiple transcripts of various lengths were detected in uterine tissues from Δ223 mutants (C). D, DNA sequencing of the Δ482 RT-PCR product demonstrated aberrant splicing between exons 2 and 4 and resulted in a frameshift creating two stop codons located immediately after the codon for Thr157. E–G, ESR1 protein was not detected in uterine tissues of the Esr1 mutant rats by western blotting (E and F) or immunofluorescence (G and H). Scale bars, 0.25 mm.
Figure 3.
Figure 3.
Effects of ESR1 disruption on postnatal growth and serum gonadotropin and sex steroid hormone levels. Adult wild-type and Esr1-null males (10 weeks of age) and adult wild-type and Esr1-null females (8 weeks of age) were weighed (A) and crown-rump length (B) and serum levels of LH (C), FSH (D), testosterone (E), and 17β-estradiol (F) measured. Sample size was ≥10 per genotype. Asterisks indicate a significant difference between the genotypes: *, P < .001; **, P < .002; ***, P < .02.
Figure 4.
Figure 4.
Effects of ESR1 disruption on the male reproductive tract. A–F, The reproductive tracts of adult wild-type and Esr1-null males (10 weeks of age) were examined, including gross appearance and weights for testes (A and B), epididymides (C and D), and seminal vesicles (E and F). Sample sizes for the organ weight measurements were ≥10 per genotype. Asterisks indicate a significant difference between the genotypes: *, P < .001. Abbreviations: Epid, epididymis; SV, seminal vesicle; wt, weight. G–L, Representative hematoxylin- and eosin-stained tissue sections of testis (G and H), caput epididymis (I and J), and cauda epididymis (K and L) from wild-type and Esr1-null males are presented. Please note the distention and atrophic changes in the seminiferous tubules and the trace numbers of sperm in the testis and epididymis of the Esr1-null rats (H, J, and L). Scale bars, 0.25 mm.
Figure 5.
Figure 5.
Effects of ESR1 disruption on the female reproductive system. A–D, Reproductive tracts of adult wild-type and Esr1-null females (8 weeks of age) were examined, including gross appearance and weights for ovaries (A and B) and uteri (C and D). Sample sizes for the organ weight measurements were ≥10 per genotype. Asterisks indicate a significant difference between the genotypes: *, P < .001. E–H, Representative hematoxylin- and eosin-stained tissue sections from ovaries (E and F) and uteri (G and H) from wild-type and Esr1-null females. I and J, Whole-mount staining of mammary glands from wild-type (I) and Esr1-null (J) females. Please note the cystic and hypoplastic features of ovaries and uteri, respectively, and the rudimentary ductal and alveolar structures in the mammary glands of Esr1-null females (F, H, and J). Scale bars, 0.25 mm.
Figure 6.
Figure 6.
Effects of ESR1 disruption on uterine responses to E2. A, Wild-type and Esr1-null female rats were ovariectomized (Ovx), rested for 2 weeks (w), and treated (red arrow) with a single dose of vehicle (oil) or E2 (40 μg/kg) and killed (Sac) 6 hours after injection. B–F, Uteri were dissected, weighed (B), and processed for quantitative RT-PCR analysis of Calb3 (C), Gadd45g (D), Igf1 (E), and Gpx2 (F). G, In a second experiment, wild-type and Esr1-null female rats were similarly prepared and then treated once daily for 3 successive days (red arrows) with oil or E2 and killed 24 hours after the last injection. H–N, Uteri were dissected, grossly inspected (I and J), weighed (H), and prepared for histological analysis and hematoxylin and eosin staining (K–N). Scale bars, 0.5 mm. Sample sizes were ≥6 per treatment. Asterisks indicate a significant difference between oil and E2 treatments: *, P < .001; **, P < .002.
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References

    1. Deroo BJ, Korach KS. Estrogen receptors and human disease. J Clin Invest. 2006;116:561–570 - PMC - PubMed
    1. Simpson ER, Misso M, Hewitt KN, et al. Estrogen–the good, the bad, and the unexpected. Endocr Rev. 2005;26:322–330 - PubMed
    1. Simpson ER, Jones ME. 2006 Of mice and men: the many guises of estrogens. Ernst Schering Found Symp Proc. 2006;1:45–67 - PubMed
    1. Adashi EY. Endocrinology of the ovary. Hum Reprod. 1994;9:815–827 - PubMed
    1. Simpson ER, Clyne C, Speed C, Rubin G, Bulun S. Tissue-specific estrogen biosynthesis and metabolism. Ann N Y Acad Sci. 2001;949:58–67 - PubMed

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