Marie-Louise Fontaine

High level production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland

The 5′ flanking region (6.3 kb) of the rabbit WAP (rWAP) gene possesses important regulatory elements. This region was linked to the human growth hormone (hGH) structural gene in order to target transgene expression to the mammary gland. Thirteen lines of transgenic mice were produced. Milk could be collected from six lines of transgenic mice. In five of them, hGH was present in the milk at high concentrations ranging from 4 to 22 mg ml−1. hGH produced by the mammary gland comigrated with hGH of human origin. It was biologically active, and through its prolactin-like activity induced lactogenesis when introduced into mammary culture media. Two of these mouse lines were studied further. hGH mRNA was only detected in the mammary gland during lactation. In the seven other transgenic lines, hGH was present in the blood of cyclic females. The prolactin-like effect of hGH in these mice probably induced female sterility, and milk could, therefore not be obtained. In two lines studied in more detail, the mammary gland was the main organ producing hGH, even in cyclic mice. Low ectopic expression was detected in other organs which varied from one line to the other. This was probably due to the influence on the transgene of the site of integration into the mouse genome. In the 13 lines studied, high mammary-specific hGH expression was not correlated to the transgene copy number. The rWAP-hGH construct thus did not behave as an independent unit of transcription. However, it can be concluded that the 6.3 kb flanking region of the rWAP gene contains regulatory elements responsible for the strong mammary-specific expression of hGH transgene, and that it is a good candidate to control high levels of foreign protein gene expression in the mammary gland of lactating transgenic animals.

Structure of the gene encoding rabbit β-casein

The entire rabbit beta-casein-encoding gene and 400 bp upstream were sequenced. Eight introns, located essentially at a position similar to the corresponding gene in other species, were found. Strong homology with several casein-encoding genes from rabbit and from other species was observed in the upstream region of the gene. Repeated sequences of unknown function were also located within introns.

Structure of the gene encoding rabbit

The complete nucleotide sequence of the entire rabbit alpha s1-casein-encoding gene Aslca and its flanking regions was determined. These data represent the first complete primary sequence of an Aslca gene. The gene consists of 19 exons spread over 16 kb. Highly conserved sequences were found between this gene and other casein-encoding genes mainly upstream from the gene from position -180 to -10. Several repeated interspersed elements of unknown function were also identified within introns.

Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes

The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone.

Structure of the rabbit αs1- and β-casein gene cluster, assignment to chromosome 15 and expression of the αs1-casein gene in HC11 cells

Abstract Several casein ( CSN ) genes ( CSN1 , 2 , 10 and αs2-CSN ) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN /BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN /BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN /BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN /BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.

Interactions between the rabbit CSN1 gene and the nuclear matrix of stably transfected HC11 mammary epithelial cells vary with its level of expression.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.