Four Core Genotypes mouse model: localization of the Sry transgene and bioassay for testicular hormone levels

Background The “four core genotypes” (FCG) mouse model has emerged as a major model testing if sex differences in phenotypes are caused by sex chromosome complement (XX vs. XY) or gonadal hormones or both. The model involves deletion of the testis-determining gene Sry from the Y chromosome and insertion of an Sry transgene onto an autosome. It produces XX and XY mice with testes, and XX and XY mice with ovaries, so that XX and XY mice with the same type of gonad can be compared to assess phenotypic effects of sex chromosome complement in cells and tissues. Findings We used PCR to amplify the Sry transgene and adjacent genomic sequences, to resolve the location of the Sry transgene to chromosome 3 and confirmed this location by fluorescence in situ hybridization (FISH) of the Sry construct to metaphase chromosomes. Using quantitative PCR, we estimate that 12–14 copies of the transgene were inserted. The anogenital distance (AGD) of FCG pups at 27–29 days after birth was not different in XX vs. XY males, or XX vs. XY females, suggesting that differences between XX and XY mice with the same type of gonad are not caused by difference in prenatal androgen levels. Conclusion The Sry transgene in FCG mice is present in multiple copies at one locus on chromosome 3, which does not interrupt known genes. XX and XY mice with the same type of gonad do not show evidence of different androgen levels prenatally.

The four core genotypes (FCG) mouse model has the advantage of separating two major factors that cause phenotypic sex differences: sex chromosome complement (XX vs. XY) and gonadal hormones [1][2][3][4][5][6][7][8][9][10]. The FCG model was established by combining two mutations in the same mouse line: deletion of the Sry gene from the Y chromosome (producing the Y − chromosome), and insertion of an Sry transgene onto an autosome [11,12]. Four genotypes are produced: XX mice with and without the Sry transgene, (XXSry, XX), and XY − mice with and without the Sry transgene (XY − Sry, XY − ). Comparing XX and XY mice of the same gonadal type allows the measurement of the effect of sex chromosome complement (XX vs. XY) on traits in a similar hormonal environment. The Sry transgene has been used in over 60 primary literature articles (Table 1), and the FCG model is available commercially (Jackson Laboratory, Bar Harbor ME, strain 010905, B6.Cg-Tg(Sry)2Ei Sry < dl1Rlb>/ArnoJ). Here we report the location and number of copies of the Sry transgene.
An important issue is whether XX and XY FCG mice with the same type of gonad experience different levels of gonadal hormones, which therefore might confound the effects of sex chromosome complement (XX vs. XY). Previous studies have not detected differences in the levels of testosterone in XX vs. XY adult males, or in estradiol in XX vs. XY females groups [33,38,47,74]; R. Schafer, personal communication), but possible differences in levels of prenatal hormones have not been assessed. Here we measured anogenital distance postnatally. Because androgens secreted prenatally by the testes cause the AGD to be larger in mice with testes than in those with ovaries [75,76], AGD is considered an excellent bioassay for the prenatal levels of androgens. These effects of androgens cause permanent sex differences in AGD, and are classified as "organizational" effects of gonadal hormones.

Methods
To identify the Sry transgene location, we first screened the DNA sequences flanking the transgene using inverted PCR [77] and vectorette PCR [78]. Amplified PCR fragments of the boundaries were sequenced, and their specificities were confirmed by PCR using 6 and 10 pairs of transgene-specific and flanking region primers on each end, using DNA from C57BL/6 FCG mice as templates.
To evaluate the influence of the Sry transgene on genes in the vicinity of the transgene, we analyzed the FCG and WT liver microarray expression datasets (GSE13264, GSE13265) [54]. Those comparable datasets were from C57BL/6J background, using the same microarray platform in the same lab. One dataset allows measuring changes in gene expression caused by the Sry transgene in gonadectomized FCG mice (using a 2-way ANOVA with factors of sex chromosome complement (XX vs. XY) and Sry transgene (present vs. absent). The other dataset compares gonadectomized WT males and females, allowing measurement of the effects of the endogenous Sry gene on the Y chromosome (one-way ANOVA). The strain origin of the Y chromosome differed in the two datasets. We report both the p-values of the ANOVAs (non-stringent analysis without correction for multiple testing), as well as more conservative False Discovery Rate p-values [79] (Table 2).

Findings
The inverted PCR and vectorette PCR methods indicated that DNA sequences flanking the transgene represent part of a repetitive motif that is found at 210-388 genomic locations (http://www.ensembl.org, Release 73). Figure 1A and B show the transgene-specific PCR amplification between Sry transgene sequence and the surrounding repetitive sequence. These were not informative for mapping the transgene in the genome, but some DNA fragments from vectorette PCR suggested that the transgene was integrated into the motif within Chr3. This conclusion was confirmed by amplification with Chr3-specific primer a and transgene-specific primer c ( Figure 1C and D). The Sry transgene integration site was at Chr3 70673749-70673824 bp ( Figure 1E, based on Ensembl Release 73), and involved deletion of 74 bp of Chr3 during integration. The integration did not interrupt any known protein coding genes or pseudogenes ( Table 3). The gene closest to the integration site is the Gm10780 pseudogene, 15 kb distant from the transgene.
To assess if the transgene affected gene expression nearby, we compared expression of 22 probes in liver in FCG mice with and without the transgene ( Table 2). Most nearby genes showed no effect of the transgene. In a few cases, expression was affected by Sry, which could have been a local effect or one mediated by testicular secretions downstream of Sry. To control for hormonally-induced changes in gene expression, we compared expression of the same genes in WT males (with endogenous Sry) vs. females using published microarray gene profiling. Two genes, Lxn and Ppid, show evidence of regulation by the  The Sry transgene band in genomic DNA from FCG was stronger than in WT ( Figure 1F), suggesting that the transgene was concatemerized during integration at this site. The number of copies of the transgene was estimated with quantitative genomic PCR (not shown) to be 12-14. The Sry transgene probe was co-localized with the Chr3 paint in metaphase spreads from FCG mice ( Figure 2).
AGD was found to differ in mice with testes vs. ovaries (Figure 3), but not in XX and XY mice of the same gonadal sex (Figure 3). A two-way ANOVA showed a significant main effect of sex (F(1,146) = 223, p < 0.00001), but no effect of sex chromosome complement (XX vs. XY, F(1,146) = 0.03, p = 0.87) and no significant interaction (F(1,146) = 0.67, p = 0.42).

Discussion
The goal of transgenic insertion is to achieve normal levels of expression of the transgene without influencing other genes because of interruption of coding or regulatory regions in the genome. Transgenic models are often useful even when this goal is not achieved in every respect. In the FCG model, mice with Sry are similar to WT males in Table 2 Expression of Chr3 genes near the Sry transgene    numerous traits, but differ for a few other traits, including higher expression of Sry, indicating that the transgene effect is similar but not identical to endogenous Sry [1,74].
Here we show that insertion of Sry onto Chr3 does not disrupt any known coding sequence. Moreover, analysis of gene expression suggests that two genes near Sry may also be altered by the transgenic insertion, as judged by expression levels in liver. Further analysis is required to determine if local genes are affected by the transgene. The FCG model has the advantage of comparing the effects of sex chromosome complement (XX vs. XY) in mice with and without the transgene. The FCG model has been useful for discovering numerous traits that are influenced by sex chromosome complement, which are independent of the presence of the transgene or have been confirmed by analysis of non-transgenic mouse models that vary sex chromosome complement [25][26][27][28]64,80]. The concatemeric insertion of 12-14 copies of a transgene at one site is not unexpected, and in the present case is associated with higher than normal expression of Sry in FCG than WT mice [74]. The greater AGD in mice with testes is expected from previous studies that demonstrate that AGD is influenced by the level of prenatal androgens. The present data offer no support for the hypothesis that the levels of androgens secreted prenatally, when AGD is determined, differ in XX and XY mice with the same type of gonad. For example, there was no masculinization of AGD of XY females relative to XX females. That result argues against the idea that XX vs. XY differences observed in numerous tissues are a result of differences in levels of prenatal androgens.
The present results contribute to the understanding of the FCG model which is used increasingly to discriminate effects of sex chromosome complement and gonadal effects on sexually dimorphic non-gonadal phenotypes ( Table 1).

Competing interests
The authors declare that they have no competing interests.
Authors' contributions YI and APA designed the study and wrote the paper. YI designed and screened the DNA sequences of Sry transgene integration site, sequenced, and confirmed it by PCR. KK and SD contributed to screening. RM measured the anogenital distance in FCG mice. RO and JB confirmed the Sry transgene chromosomal location via FISH. All authors read and approved the final manuscript. Figure 3 Anogenital distance measurement of FCG mice. Asterisk shows the significant effect of sex (two way ANOVA, p < 0.00001).