Generation and identification of a conditional knockout allele for the PSMD11 gene in mice

Background Our previous study have shown that the PSMD11 protein was an important survival factor for cancer cells except for its key role in regulation of assembly and activity of the 26S proteasome. To further investigate the role of PSMD11 in carcinogenesis, we constructed a conditional exon 5 floxed allele of PSMD11 (PSMD11flx) in mice. Results It was found that homozygous PSMD11 flx/flx mice showed normal and exhibited a normal life span and fertility, and showed roughly equivalent expression of PSMD11 in various tissues, suggesting that the floxed allele maintained the wild-type function. Cre recombinase could induce efficient knockout of the floxed PSMD11 allele both in vitro and in vivo. Mice with constitutive single allele deletion of PSMD11 derived from intercrossing between PSMD11flx/flx and CMV-Cre mice were all viable and fertile, and showed apparent growth retardation, suggesting that PSMD11 played a significant role in the development of mice pre- or postnatally. No whole-body PSMD11 deficient embryos (PSMD11−/−) were identified in E7.5–8.5 embryos in uteros, indicating that double allele knockout of PSMD11 leads to early embryonic lethality. To avoid embryonic lethality produced by whole-body PSMD11 deletion, we further developed conditional PSMD11 global knockout mice with genotype Flp;FSF-R26CAG − CreERT2/+; PSMD11flx/flx, and demonstrated that PSMD11 could be depleted in a temporal and tissue-specific manner. Meanwhile, it was found that depletion of PSMD11 could induce massive apoptosis in MEFs. Conclusions In summary, our data demonstrated that we have successfully generated a conditional knockout allele of PSMD11 in mice, and found that PSMD11 played a key role in early and postnatal development in mice, the PSMD11 flx/flx mice will be an invaluable tool to explore the functions of PSMD11 in development and diseases.

A floxed allele of the androgen receptor gene causes hyperandrogenization in male mice

We previously generated a conditional floxed mouse line to study androgen action, in which exon 3 of the androgen receptor ( AR) gene is flanked by loxP sites, with the neomycin resistance gene present in intron 3. Deletion of exon 3 in global AR knockout mice causes androgen insensitivity syndrome, characterized by genotypic males lacking normal masculinization. We now report that male mice carrying the floxed allele (ARlox) have the reverse phenotype, termed hyperandrogenization. ARlox mice have increased mass of androgen-dependent tissues, including kidney, ( P < 0.001), seminal vesicle ( P < 0.001), levator ani muscle ( P = 0.001), and heart ( P < 0.05). Serum testosterone is not significantly different. Testis mass is normal, histology shows normal spermatogenesis, and ARlox males are fertile. ARlox males also have normal AR mRNA levels in kidney, brain, levator ani, liver, and testis. This study reaffirms the need to investigate the potential phenotypic effects of floxed alleles in the absence of cre in tissue-specific knockout studies. In addition, this androgen hypersensitivity model may be useful to further investigate the effects of subtle perturbations of androgen action in a range of androgen-responsive systems in the male.

GA Binding Protein (GABP) Is Required for Myeloid Cell Development and Differentiation.

GABPα is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPα binds to DNA through its ets domain and forms a multimeric complex by recruiting its partner, GABPβ, which contains the transactivation domain. GABPα is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPβ to DNA. We cloned GABPα from a murine genomic BAC library and prepared a targeting vector in which the GABPα ets domain is flanked by loxP recombination sites (floxed allele, designated fl). Mice that bear one intact (and one disrupted copy) of GABPα, i.e. hemizygous mice, are phenotypically normal. Intercrossing of hemizygous mice yielded no nullizygous mice, indicating that homozygous loss of GABPα causes an embryonic lethal defect. To determine the effect of GABPα deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to mice that bear the interferon-responsive Mx1-Cre transgene, which express Cre in response to injection of the synthetic polynucleotide, poly I-C. Bone marrow cells underwent efficient deletion of GABPα following poly I-C injection; in contrast, other somatic tissues did not efficiently delete the floxed allele. Bone marrow, peripheral blood, and other tissues were examined for cellular morphology and flow cytometry. We compared mice that lack GABPα in bone marrow (i.e. fl/fl Mx1-Cre mice injected with poly I-C) to littermate controls (i.e. fl/fl mice injected with poly I-C). Mice that lack GABPα exhibited a striking and statistically significant decrease in granulocytes and monocytes in bone marrow and peripheral blood, compared with controls; in contrast, there was an increase in erythroid cells in GABPα null bone marrow. This indicates that the loss of GABPα has lineage-specific effects on myeloid cell development. Morphologic analysis indicates that mice which lack GABPα possess more immature granulocytes compared to control mice. Thus, GABP disruption causes a striking loss of myeloid cells in the bone marrow and peripheral blood of mice in a lineage-specific manner. Furthermore, the maturation block of murine granulocytes that is caused by GABPα disruption demonstrates the crucial role of GABP in myeloid differentiation.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.