The ABCB1-1Delta mutation is not responsible for subchronic neurotoxicity seen in dogs of non-collie breeds following macrocyclic lactone treatment for generalized demodicosis.

P-glycoprotein (P-gp), encoded by the multiple drug resistance gene ABCB1 (also known as MDR1), is an integral component of the blood brain barrier crucial in limiting drug uptake into the central nervous system. Altered expression or function of P-gp, as seen in dogs of the collie lineage homozygous for the nt228(del4) mutation of the ABCB1 gene (ABCB1-1Delta), can result in potentially fatal neurotoxicosis, especially following administration of systemic macrocyclic lactones (SML). Occasionally, dogs from unrelated breeds develop subchronic signs of neurotoxicity when receiving SML to treat generalized demodicosis. It is possible that these dogs are heterozygous carriers of the ABCB1-1Delta mutation, resulting in decreased P-gp activity and central neurotoxicosis. Cheek swabs were collected from 28 dogs with generalized demodicosis that had shown subchronic signs of neurotoxicity following daily oral administration of ivermectin or other SML. Ten of these animals received concurrent systemic treatment with other confirmed or putative P-gp substrates. After DNA extraction, the relevant portion of the ABCB1 gene was amplified by polymerase chain reaction, and sequenced. Twenty-seven dogs were homozygous normal while one dog was heterozygous for the ABCB1-1Delta mutation. Therefore, with the exception of one dog, the observed neurotoxicity could not be attributed to the ABCB1-1Delta mutation. Possible explanations for the adverse reactions observed include pharmacological interactions (administration of SML with other P-gp substrates or inhibitors), excessively high doses, polymorphisms in P-gp expression, uncharacterized mutations in the ABCB1 gene or in another gene, or phenomena unrelated to the SML-P-gp interaction.

Porcine SRY promoter is a target for steroidogenic factor 1.

To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated a 4.5-kilobase (kb) pig SRY 5' flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at -661 base pairs (bps) 5' from the translation initiation site. Deletion studies of the SRY 5' flanking sequences in PGR 9E11 cells demonstrated that -1.4 kb of 5' flanking sequences retained full transcriptional activity compared with the -4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, because deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the -1.4 kb fragment identified two potential binding sites for SF-1, at -1369 and at -290 from the ATG. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse SF-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at -1369 bp and at -290 bp, and that the site at -1369 bp is quantitatively the most important.

Conservation of the function of DMRT1 regulatory sequences in mammalian sex differentiation.

Among genes involved in sex determination and differentiation, DMRT1 is the only one characterized to date containing a domain (the DM domain) that is conserved between phyla. To study DMRT1 transcriptional regulation within mammalian phyla, we generated transgenic mice that express green fluorescent protein (GFP) or Cre-recombinase (Cre) under the control of 2.6 kb of pig DMRT1 5' flanking sequences (pDMRT1p-GFP and pDMRT1p-Cre, respectively). Within the pDMRT1p-GFP positive mice, GFP expression was observed in the XY genital ridge by embryonic day 11.5 (e11.5) and remained detectable during testis embryonic development to birth. GFP expression was restricted within testis cords as soon as cords were detectable. No fluorescence was observed in developing ovaries, although more sensitive RT-PCR analysis revealed transgene expression in embryonic ovaries from e13.5 to e15.5. RT-PCR performed on fluorescent activated cell sorter (FACS)-purified GFP cells from e14.5, e17.5, and e19.5 developing testis showed that GFP expression was restricted to cells expressing the endogenous mouse Dmrt1. GFP cells also expressed Mis and Oct4, showing that the transgene is expressed in both Sertoli cell and germ cell compartments. In postnatal testis, transgene expression was detectable by GFP fluorescence from P0 to P21 in mice heterozygous for the transgene and through adulthood in mice homozygous for the transgene. In pDMRT1p-Cre positive mice, Cre expression was detected within the genital ridges of both XY and XX embryos. We conclude that DMRT1 regulatory mechanisms during sexual differentiation are functionally conserved across mammalian evolution. The transgenic mouse lines described should provide useful marker systems for studies involving Dmrt1 gene expression during sex differentiation.

The porcine SRY promoter is transactivated within a male genital ridge environment.

In mammals the SRY gene functions as a dominant genetic switch for testis determination (Gubbay et al.: Nature 346:1128-1135, 1990; Koopman et al.: Nature 351:117-121, 1991; Sinclair et al.: Nature 346:240-244, 1990). To study SRY transcriptional regulation within an evolutionary context, we have generated transgenic mice that express green fluorescent protein (GFP) under the control of 4.5 kb of pig SRY 5' flanking sequences (pSRYp-GFP). Autofluorescence was observed in the genital ridges of e11.5 male embryos (18-21 tail somites), and by e12.5 (27 tail somites) autofluorescence was observed within the testes cords. The expression of the transgene did not display the abrupt termination characteristic of endogenous mouse SRY, but rather showed a gradual reduction in expression characteristic of human, pig and sheep SRY. Surprisingly, no autofluorescence was observed in normal XX genital ridges, although more sensitive RT-PCR analysis detected transgene transcription. When the transgene was bred into a constitutively male line of mice (Odsex; Bishop et al.: Nat Genet 26:490-494, 2000), autofluorescence was visible in genital ridges of XX animals, in the genetic absence of Sry protein. Via RT-PCR analysis, purified autofluorescent cells from e12.5 gonadal ridges expressed mouse SRY but not Oct4 transcripts, whereas autofluorescent cells from e14.5 gonadal ridges expressed MIS but not Oct4 transcripts, in each case consistent with a pre-Sertoli cell phenotype. In vitro expression studies performed in CV-1 cells demonstrated that pig SOX9 cDNA transactivated the pig SRY promoter but that pig SRY cDNA did not. When a SOX9 potential binding site identified at -205 of the pig SRY 5' flanking sequences was mutated, the SOX9 transactivation effect was reduced by 70%. This site is conserved in the 5' flanking sequences of bovine and human SRY genes but not in the mouse gene. Gel retardation assays using this binding site showed specific binding to SOX9-enriched nuclear extracts that was competed by excess unlabelled binding site but not by mutated binding site. We suggest that pig SRY gene is responsive to a testicular environment and propose a model of feedback amplification of pig SRY transcription by SOX9.