Isabelle Daneau

Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation.

We have generated complete complementary DNA (cDNA) sequences for the porcine steroidogenic acute regulatory protein (StAR) gene, using a combination of genomic PCR amplification and reverse transcription-PCR amplification of pig ovarian cDNA. Porcine StAR cDNA consists of 855 bp and shares 90.2%, 87.3%, 84.3%, and 83.9% homologies with bovine, human, mouse, and rat StAR cDNA at the nucleotide level, and 89.1%, 88.8%, 86.7%, and 86.3% homologies with bovine, human, mouse, and rat StAR protein at the deduced amino acid level. Northern analysis of porcine StAR showed that it is expressed in adult and fetal steroidogenic tissues, including adult testes and ovaries and adult adrenal glands as well as steroidogenic tissues of pregnancy, including developing fetal testes, corpus luteum, and pregnancy, but not the fetal ovary. Major hybridizing bands of 1.8 and 1.1 kilobases were demonstrated. In contrast to human StAR, porcine StAR was not expressed in adult or fetal kidneys. Expression of porcine StAR by the p...

Porcine SRY Promoter Is a Target for Steroidogenic Factor 1

Abstract 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.

The ABCB1-1Δ 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.

Genetic manipulation of sex differentiation and phenotype in domestic animals

In mammals, a gene based sex determination system ensures that approximately 50% of offspring will be of the male sex and 50% will be of the female sex. In domestic animal production systems, this ratio is not always ideal. Recent advances in our understanding of the molecular biology of sex determination and differentiation, as well as in the control of gene expression and the direct modification of animal genomes, allows us to consider methods for the direct genetic manipulation of sexual phenotype.