Generation and validation of a mouse line with a floxed SRC-3/AIB1 allele for conditional knockout.

The steroid receptor coactivator-3 (SRC-3), also known as AIB1, ACTR, p/CIP and NCOA3, is a transcriptional coactivator for nuclear receptors and certain other transcription factors. SRC-3 is widely expressed and plays important physiological functions and pathogenic roles in breast and prostate cancers. SRC-3 knockout (SRC-3-/-) mice display genetic background-dependent embryonic lethality and multiple local and systemic abnormalities. Since both the partial lethality and the systemic effects caused by global SRC-3 knockout interfere with downstream investigation of tissue-specific function of SRC-3, we have generated floxed SRC-3 (SRC-3f/f) mice with conditional alleles carrying loxP sites in introns 10 and 12 by a gene-targeting strategy. The two SRC-3f/f mouse lines (A and B) are indistinguishable from wild type mice. To test if deletion of the floxed exons 11 and 12 for SRC-3 nuclear receptor interaction domains and disruption of its downstream sequence for transcriptional activation domains would inactivate SRC-3 function, SRC-3f/f mice were crossbred with EIIa-Cre mice to generate SRC-3d/d mice with germ line deletion of the floxed SRC-3 gene. Both lines of SRC-3d/d mice exhibited growth retardation and low IGF-I levels, which was similar to that observed in SRC-3-/- mice. The line A SRC-3d/d mice showed normal viability, while line B SRC-3d/d mice showed partial lethality similar to SRC-3-/- mice, probably due to variable distributions of genetic background during breeding. These results demonstrate that the floxed SRC-3 mouse lines have been successfully established. These mice will be useful for investigating the cell type- and developmental stage-specific functions of SRC-3.

SRC-3 is expressed in brain, vascular endothelial and smooth muscle cells, intestinal smooth muscle cells, mammary gland epithelium, oocyte, prostate stromal and basal cells, mammary gland and prostate tumor cells, Sertoli cells in the testis and liver cells (16,17). SRC-3 knockout (SRC-3 -/-) mice have been previ-ously generated by conventional gene-targeting strategies to explore the in vivo function of SRC-3 (16,18). Global inactivation of the SRC-3 gene in mice causes partial embryonic lethality depending on strain genetic backgrounds ((16) and unpublished data) and growth retardation accompanied with low IGF-I and IGF-binding protein 3 (IGFBP-3) levels, delays pubertal development, reduces female reproductive function and mammary gland growth accompanied with low estrogen levels (16,18,19), decreases estrogen-dependent vascular protection (20), inhibits adipogenesis (21) and increases inflammatory responses (22). These findings indicate that SRC-3 is widely expressed, and it regulates physiological functions through both its cell autonomous cellular actions and its systemic effects on hormones such as IGF-I and estrogen.
Although SRC-3 -/mice have provided valuable information regarding fundamental genetic functions of SRC-3, the diverse and systemic effects of global SRC-3 deficiency have hindered our understanding of tissue-and cell type-specific and developmental stage-specific functions of SRC-3. In order to circumvent these limitations, we have generated and validated mouse lines carrying a floxed SRC-3 allele that is appropriate for Cre-mediated tissue-specific inactivation of the SRC-3 gene.

DNA cloning, embryonic stem (ES) cell culture and gene targeting
The mouse SRC-3 genomic DNA was obtained from phage clones containing large fragments of the SRC-3 genomic DNA from the 129SvEv mouse genome (16,23). A 5.65-kb DNA fragment for the 5' targeting arm was taken from the SRC-3 genomic DNA in a phage clone by Apa I and Sca I co-digestion and subcloned into the Pac I/Asc I sites of the pFRT-LoxP plasmid. A 1.9-kb SRC-3 DNA fragment to be floxed was taken from the same clone by Sca I and Mfe I co-digestion and subcloned into the Not I and Kpn I sites of the same plasmid. A 5-kb SRC-3 DNA fragment for the 3' targeting arm was taken from another SRC-3 genomic DNA clone by Mfe I and Bmg BI co-digestion and subcloned into the Xho I and Bam HI sites of the same plasmid for finishing construction of the targeting vector (Fig. 1). The vector was linearized by Xho I (inserted after the 5' arm) digestion and transfected into the TC-1 129SvEv mouse ES cells (24) by electroporation. Transfected ES cells were cultured in selection medium with G418 (Geneticin) and FIAU (1-(2-deoxy-2-fluoro-beta-D-arabino furanosyl)-5-iodouracil) and survived clones were isolated for Southern blot analysis as described previously (16,25). The 5' probe DNA was taken from the SRC-3 genomic DNA clone by Sal I and Apa I digestion, which was located upstream at the 5' targeting arm (Fig. 1). The 3' probe DNA was taken from the SRC-3 genomic DNA clone by Bmg BI and Kpn I digestion, which was located downstream at the 3' targeting arm (Fig. 1). Southern blot analysis also was preformed with a neo probe to exclude clones with any random integrations of the targeting vector DNA. After DNA is digested with Sca I and Sal I, the 5' probe should detect a 9.6-kb band for WT allele and a 11.5-kb band for the targeted allele as indicated. After digested with Bam HI and Kpn I, the 3' probe should detect a 6.2-kb band for the WT allele and a 7.5-kb band for the targeted allele as indicated. The floxed allele can be obtained after excision of the Frt-flanked neo cassette through crossing to FLPeR transgenic mice. The P1, P2, P5 and P3 primers for PCR-based genotype analyses are indicated (arrowheads). The structure of the deleted allele obtained after Cre-mediated excision of the floxed region is also sketched.

Microinjection and mouse breeding
The correctly targeted ES clones were microinjected into C57/BL6 morulas to generate chemeric founder mice. The male chimeric mice were kept for breeding with female C57BL/6 mice to test germ line transmission of the targeted SRC-3 locus. Next, the male chimeric mice with proven germ line transmission ability were bred with the 129SvEv FLPeR mice (Stock #003946, Jackson Lab) (26) to remove the Frt-flanked neo expression cassette (Fig. 1). Finally, the FLPeR allele was excluded in incoming generations and the floxed SRC-3 allele was excised in germ line by breeding with the C57BL/6 EIIa-Cre transgenic mice (Stock #003724, Jackson Lab) (27).

Body weight and IGF-1 measurements
Male mice were weighed at 4, 8 and 12 weeks of age. Blood samples were collected from 12-week-old mice and serum samples were prepared and frozen at -80°C until analysis. Serum IGF-1 concentrations were measured using an ELISA kit (DSL-10-29200, Diagnostic Systems Lab.) by following the manufacturer's instruction. Results are presented as mean ± standard deviation. Statistical analysis was performed using unpaired Student's t test with p<0.05 to be considered as significant difference.

Generation of ES cells with targeted SRC-3 allele by homologous recombination
The mouse SRC-3 gene spans about 79 kb in length and contains 23 exons, and it encodes a 160-kD protein with 1403 amino acid residues (23). SRC-3 protein contains an N-terminal basic helix-loop-helix/PAS (Per/ARNT/Sim) domain, a central nuclear receptor interaction domain with multiple LXXLL (L, leucine, X, any amino acid residue) motifs, and a C-terminal transcriptional activation domain (1,28). To obtain a floxed SRC-3 allele appropriate for Cre-mediated conditional inactivation of the SRC-3 gene, we designed and constructed a targeting vector that could knock in one loxP site into intron 10 and another loxP site into intron 12 through homologous recombination in mouse ES cells. The targeting vector contains a 5.65-kb 5' targeting arm, a floxed 1.9-kb DNA fragment spanning exons 11 and 12 and intron 11, a neo expression cassette for positive selection, a 5-kb 3' targeting arm and a tk expression cassette for negative selection (Fig. 1). The floxed exons 11 and 12 encode the nuclear receptor interaction domain of SRC-3 (23,28). In addition, the deletion of exons 11 and 12 would also cause a coding-frame shift between exons 10 and 13 and result in disruption of the C-terminal polypeptide sequence of SRC-3 (Fig. 1).
After the TC-1 mouse ES cells were electroporated with the targeting vector DNA and cultured in selection medium with G418 and FIAU, 288 survived clones were isolated and DNA samples were prepared from these clones for Southern blot screening. The 3' probe detected 10 clones with a 7.5-kb targeted allele band in addition to a 6.2-kb wild type (WT) allele band after DNA samples were digested with Bam H1 and Kpn I ( Fig. 1 and Fig. 2B). Four of these positive clones also showed correct recombination at the 5' location, exhibiting a 11.5-kb targeted allele band in addition to a 9.6-kb WT allele band on Southern blot using the 5' probe after DNA was digested with Sca I and Sal I ( Fig. 1 and Fig. 2A). A neomycin probe was also used to confirm the sole homologous integration locus of the targeting vector at the SRC-3 gene in the targeted ES cells (Fig. 2C). These results demonstrate that multiple targeted ES clones with the floxed SRC-3 allele had been successfully generated. Since this targeted allele contains an Frt-flanked neo cassette, this allele is designated as SRC-3 f-neo .

Generation of SRC-3 f/f mice
Correctly targeted ES clones were microinjected into C57BL/6 blastocysts to generate chimeric mice. Male chimeras were kept for testing germ line transmission of the SRC-3 f-neo allele by crossing with C57BL/6 female mice and by examining the coat color of their offspring. Agouti coat indicates a germ line transmission from the 129SvEv ES cell, while black coat indicates a C57BL/6 blastocyst donor origin. A total of 7 chimeric founder mice were identified to have germ line transmission capability. Two of these founder mice (lines A and B) were crossed with female FLPeR mice (26) to remove the Frt-flanked neo cassette in their pups with an SRC-3 f-neo/+ /FLPeR + genotype. In these pups, the flippase recombinase (FLP) was ubiquitously expressed and its expression resulted in excision of the Frt-flanked neo cassette in the SRC-3 f-neo allele. As shown in Fig. 3, PCR analyses detected WT and targeted alleles in FLPeR-negative SRC-3 f-neo/+ mice (Fig. 3A) and WT and floxed alleles in FLPeR-positive and neo-negative SRC-3 f/+ mice (Fig.  3B). Subsequently, SRC-3 f/+ breeding pairs were used to generate WT and SRC-3 f/f mice that were negative of both neo cassette and FLPeR. Because the normal function of the SRC-3 gene is required for somatic growth and normal IGF-I levels, we measured body growth rates and serum IGF-I levels and found no differences between WT and SRC-3 f/f littermates with a pure 129SvEv strain background (Fig. 4, A & B). These results indicate that the SRC-3 f allele has normal function identical to the SRC-3 + allele. Genotype analysis of SRC-3 f/+ mice after the neo cassette is excised by crossing to FLPeR mice. Both SRC-3 + and SRC-3 f alleles were detected by PCR using SRC3-P3/SRC3-P5 primers. Due to both loxP and Frt palindromic sequences in the floxed allele, the product from PCR using primers SRC3-P3 and SRC3-P5 produces two bands for the SRC-3 f allele and one band for the SRC-3 + allele. C. Genotype analyses of mice derived from SRC-3 f/+ and EIIa-Cre breeding pairs. Both SRC-3 + and SRC-3 f were detected by PCR using SRC3-P1 and SRC3-P2 primers as shown in panel A. The SRC-3 d allele was detected by PCR using SRC3-P1 and SRC3-P3 primers. Both SRC-3 d and SRC-3 f alleles were detected in SRC-3 f/+ /EIIa-Cre + mice due to partial excision of the floxed SRC-3 allele. D. Genotype analyses of mice derived from SRC-3 f/+ /EIIa-Cre + (SRC-3 f-or-d/+ ) breeding pairs. PCR was performed as in panel C. From this breeding, mice with germ line deletion of the floxed SRC-3 (SRC-3 d/d ) were generated and identified.

Germ-line deletion of the floxed SRC-3 allele recapitulates an SRC-3 knockout phenotype
To test whether excision of the floxed region of the SRC-3 f allele would completely inactivate the gene function, we crossed SRC-3 f/f mice with a 129SvEv strain background (both A and B lines) with EIIa-Cre transgenic mice with a C57BL/6 strain background (27) and generated SRC-3 f/+ /EIIa-Cre + mice with a mixed genetic background. In these mice, the ex-pressed Cre resulted in a partial deletion of the SRC-3 f allele and produced mosaic mice with both the deleted SRC-3 (SRC-3 d ) and the remaining SRC-3 f alleles in addition to the WT SRC-3 allele in the same mouse as determined by PCR genotyping using tail-tip DNA samples (Fig. 3C). Subsequently, SRC-3 d/+ and SRC-3 d/d mice were generated by breeding SRC-3 f/+ /EIIa-Cre + mice and their progenies (Fig. 3D) For both lines A and B, SRC-3 d/+ mice were slightly smaller than age-matched WT mice (Data not shown). However, both male and female SRC-3 d/d mice were significantly smaller than WT mice at all ages examined, including 4, 8 and 12 weeks (Fig. 4C for line A, and data for line B not shown). When measured at 12 weeks of age, SRC-3 d/d mice also had a remarkable reduction in serum IGF-I levels compared with age-matched WT mice (Fig. 4D for line A, and data for line B not shown). The growth defect and low levels of IGF-I phenotypes observed in SRC-3 d/d mice closely resemble the SRC-3 knockout mice (16). These results demonstrate that excision of the floxed region (exons 11 and 12) of the SRC-3 f allele results in a loss-of-function mutant of the SRC-3 allele.

Discussion
Since SRC-3 is identified as a transcriptional coactivator, many studies have been performed to explore its normal functions in development, metabolism, reproduction and cardiovascular system, and its pathogenetic roles in hormonally promoted breast and prostate cancers (11-13, 15, 16, 18-20, 29). Because no germ line mutation has been found to link SRC-3 to a human disease, the knowledge regarding most of the SRC-3 in vivo functions is gained from SRC-3 knockout and transgenic mice. Although these mouse models provide valuable information, their limitations of systemic effects caused by global SRC-3 deficiency have made it difficult to assign certain phenotypes and their molecular basis to a specific cell type or a specific developmental stage. In order to use the Cre-loxP system to inactivate SRC-3 in either a cell type-specific or a developmental stage-specific way, we have generated SRC-3 f/f mice using a gene targeting strategy in this study. SRC-3 f/f mice exhibit normal somatic growth and normal IGF-I levels and are indistinguishable from WT mice, indicating that insertion of the loxP sites has no effect on the SRC-3 gene function.
In SRC-3 f/f mice, the paired loxP sites are located in introns 10 and 12. The floxed exons 11 and 12 encode the essential nuclear receptor interaction domain and the deletion of these two exons also disrupts the downstream coding frame. As expected, in our testing experiments deletion of the floxed SRC-3 alleles in SRC-3 d/d mice caused postnatal growth retardation and low levels of circulating IGF-I, which are two of the major defects observed in SRC-3 knockout mice (1,16,18). These results demonstrate that Cre-mediated excision of the floxed SRC-3 allele disrupt the function of the SRC-3 gene. Intriguingly, the two lines of SRC-3 d/d mice exhibit different viability. The line A SRC-3 d/d mice have a normal viability while the line B SRC-3 d/d mice show a partial lethality. This discrepancy may be caused by uneven distributions of 129SvEv and C57BL/6 strain backgrounds in these two lines of mice during the breeding process. In agreement with this explanation, our recent studies have revealed that the lethal degrees of SRC-3 -/mice are dependent on strain background. Lesser, medium and severe degrees of lethality are found in SRC-3 -/mice with a 129SvEv, a mixed 129SvEv/C57BL/6 and a C57BL/6 strain background, respectively ((16) and unpublished results).
Taken together, we have generated and validated the SRC-3 f/f mouse line appropriate for conditional inactivation of the SRC-3 gene after crossing with a Cre mouse line of interest. The SRC-3 f/f mouse line will be useful for investigating the cell type-and developmental stage-specific functions of SRC-3 in mice.