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. 2022 Jan-Feb;24(1):102-108.
doi: 10.4103/aja.aja_38_21.

Polymerase chain reaction-based assays facilitate the breeding and study of mouse models of Klinefelter syndrome

Hai-Xia Zhang  1 , Yu-Lin Zhou  1 , Wen-Yan Xu  1 , Xiao-Lu Chen  1 , Jia-Yang Jiang  2 , Xiao-Man Zhou  1 , Zeng-Ge Wang  3 , Rong-Qin Ke  2 , Qi-Wei Guo  1
Affiliations

Polymerase chain reaction-based assays facilitate the breeding and study of mouse models of Klinefelter syndrome

Hai-Xia Zhang et al. Asian J Androl. 2022 Jan-Feb.

Abstract

Klinefelter syndrome (KS) is one of the most frequent genetic abnormalities and the leading genetic cause of nonobstructive azoospermia. The breeding and study of KS mouse models are essential to advancing our knowledge of the underlying pathological mechanism. Karyotyping and fluorescence in situ hybridization are reliable methods for identifying chromosomal contents. However, technical issues associated with these methods can decrease the efficiency of breeding KS mouse models and limit studies that require rapid identification of _target mice. To overcome these limitations, we developed three polymerase chain reaction-based assays to measure specific genetic information, including presence or absence of the sex determining region of chromosome Y (Sry), copy number of amelogenin, X-linked (Amelx), and inactive X specific transcripts (Xist) levels. Through a combined analysis of the assay results, we can infer the karyotype of _target mice. We confirmed the utility of our assays with the successful generation of KS mouse models. Our assays are rapid, inexpensive, high capacity, easy to perform, and only require small sample amounts. Therefore, they facilitate the breeding and study of KS mouse models and help advance our knowledge of the pathological mechanism underlying KS.

Keywords: 40; XXY mouse; Klinefelter syndrome; mouse model; XXY*mouse; 41.

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Conflict of interest statement

None

Figures

Figure 1
Figure 1
Breeding of KS mouse models. The mouse in the blue circle is a 40,XXY* mouse, one of the KS mouse models. The mice in the red circles are the _target mice in each generation for breeding 41,XXY mice, another KS mouse model. This figure was modified from figures in a previous study. NPX: nonpseudoautosomal region of the X chromosome; NRY: nonrecombining region of the Y chromosome; PAR: pseudoautosomal region. Sry: sex determining region of chromosome Y; Amelx: amelogenin, X-linked; Xist: inactive X specific transcripts; KS: Klinefelter syndrome.
Figure 2
Figure 2
Genetic information of the mouse breeds obtained during the breeding of Klinefelter syndrome mouse models. +: presence of Sry or high Xist RNA transcripts; −: absence of Sry or low/no Xist RNA transcripts. Pink labels indicate the _target mice in each generation for the breeding of 41,XXY mice. Green labels indicate the assays required to identify the _target mice in each generation. For example, in the second generation, each mouse was first tested for Sry, and those negative for Sry were further tested for Xist RNA transcript levels. Mice with low/no Xist RNA transcripts (negative results in both assays) were identified as 40,XY*X mice. Sry: sex determining region of chromosome Y; Amelx: amelogenin, X-linked; Xist: inactive X specific transcripts.
Figure 3
Figure 3
Polymerase chain reaction-based assays for the breeding of Klinefelter syndrome mouse models. (a) Melting profiles for detecting Sry using different amounts of DNA template. (b) Assays to quantify Amelx using different amounts of DNA template. (c) Establishment of a cutoff value for Amelx quantification. (d) Assays for evaluating Xist RNA transcript levels using different amounts of RNA template. (e) Establishment of a cutoff value for Xist RNA quantification. Each sample was tested in triplicate, and the Cq value of each sample is the mean Cq value of three replicates. NTC: no template control. Sry: sex determining region of chromosome Y; Amelx: amelogenin, X-linked; Xist: inactive X specific transcripts; Actb: actin, beta; Cq: quantification cycle.
Figure 4
Figure 4
Karyotypes of the _target mice for breeding Klinefelter syndrome mouse models. (a) A 40,XXY* mouse from the second generation. The arrow indicates the XY* chromosome. (b) A 40,XY*X mouse from the second generation. The arrow indicates the Y*X chromosome. (c) A 41,XYY*X mouse from the third generation. The arrow indicates the Y*X chromosome. (d) A 41,XXY mouse from the fourth generation.
Figure 5
Figure 5
Testicular phenotypes of Klinefelter syndrome mouse models. (a) Appearance, (b) anatomic analysis, and (c) histologic analysis of the testes in a 40,XXY* mouse and 40,XY* littermate. (d) Appearance, (e) anatomic analysis, and (f) histologic analysis of the testes in a 41,XXY mouse and 40,XY littermate. The arrows indicate the position of the testes. As expected, 40,XXY* and 41,XXY mice presented small, firm testes. Moreover, the tubule diameters of 40,XXY* and 41,XXY mice were smaller than those of their reference littermates, germ cells were absent, and only Sertoli cells were observed within the tubules.
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References

    1. Klinefelter HF, Reifenstein EC, Albright F. Syndrome characterized by gynecomastia aspermatogenesis without a-leydigism and increased excretion of follicle stimulating hormone. J Clin Endocrinol Metab. 1942;2:615–27.
    1. Berglund A, Stochholm K, Gravholt CH. The epidemiology of sex chromosome abnormalities. Am J Med Genet C Semin Med Genet. 2020;184:202–15. - PubMed
    1. Forti G, Corona G, Vignozzi L, Krausz C, Maggi M. Klinefelter's syndrome: a clinical and therapeutical update. Sex Dev. 2010;4:249–58. - PubMed
    1. Gravholt CH, Chang S, Wallentin M, Fedder J, Moore P, et al. Klinefelter syndrome: integrating genetics, neuropsychology, and endocrinology. Endocr Rev. 2018;39:389–423. - PubMed
    1. Skakkebaek A, Gravholt CH, Chang S, Moore PJ, Wallentin M. Psychological functioning, brain morphology, and functional neuroimaging in Klinefelter syndrome. Am J Med Genet C Semin Med Genet. 2020;184:506–17. - PubMed
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