Marie-Georges Stinnakre

The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice

Various combinations of promoters, introns and transcription terminators were used to drive the expression of bovine growth hormone (bGH) cDNA in different cell types. In constructs containing the human cytomegalovirus (hCMV) promoter and the SV40 late genes terminator, the intron from SV40 genes (VP1) was much more efficient, than the intron from the early genes (t). The synthetic intron SIS generated by the association of an adenovirus splice donor and an immunoglobulin G splice acceptor showed the highest activity. The respective potency of these introns was similar in several mammalian (CHO, HC11 and COS) and fish (TO2 and EPC) cells. The rabbit whey acidic protein (WAP) gene promoter was highly efficient to drive the expression of bGH gene in the HC11 mammary cell lines. In contrast, the bGH cDNA under the control of the same promoter was much less efficiently expressed when the SV40 VP1 intron and transcription terminator were used. The rabbit WAP gene and the human GH gene terminators did not or only moderately enhanced the expression of the construct WAP bGH cDNA. Introduction of a promoter sequence from the mouse mammary tumor virus (MMTV) LTR in the VP1 intron increased very significantly the expression of the WAP bGH cDNA. Although several of these vectors showed high potency when expressed stably in HC11 cells, all of them were only moderately efficient in transgenic mice. These data indicate that the VP1 and the SIS introns may be used to express foreign cDNAs with good efficiency in different cell types. The addition of an enhancer within an intron may still reinforce its efficiency. However, transfection experiments, even when stable expression is carried out, are poorly predictive of the potential efficiency of a vector in transgenic animals.

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.

Expression analysis of ruminant α-lactalbumin in transgenic mice: Developmental regulation and general location of important cis-regulatory elements

The bovine α‐lactalbumin transgene with 750 bp and 336 bp of the 5′ and 3′ flanking region, respectively, is developmentally regulated as its endogenous counterpart in transgenic mice. Comparative expression analysis of three 5′‐shortened constructs suggests that the region −4771/−220 contains important cis‐acting transcriptional elements. The level of expression of a long caprine α‐lactalbumin transgene encompassing 8.5 kb and 9.5 kb of the 5′ and 3′ flanking region, respectively, was higher but still unrelated to the copy number. Expression of the transgenes and of endogenous milk‐protein genes was tissue‐specific. In contrast with a recent report, only low amounts of the relevant mRNA were detected in some skin samples, which suggests a possible contamination by mammary tissue.

The bovine α-lactalbumin promoter directs expression of ovine trophoblast interferon in the mammary gland of transgenic mice

A hybrid construct derived from ovine trophoblastin cDNA and bovine α‐lactalbumin‐encoding gene, was injected into the pronuclei of mouse eggs. In one of the resulting transgenic mouse lines, expression of the hybrid construct was detected and found to be limited to the mammary gland of lactating females which secreted active ovine trophoblastin. This strongly suggests that important cis‐acting DNA sequences involved in tissue‐specific expression of the bovine gene are located within the second half of the 3′ untranslated region, or/and the proximal 5′and 3′ regions flanking the transcriptional unit.

Introduction of a Proximal Stat5 Site in the Murine α-Lactalbumin Promoter Induces Prolactin Dependency In Vitro and Improves Expression Frequency In Vivo

In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position −70 on a 560 bp murine α-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.

Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine beta-lactoglobulin gene that confer locus commitment to heterogeneous tissues.

In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine β-lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus

The synthesis of a recombinant protein in milk of transgenic mice

Abstract Micro-organisms and lower euraryotes can synthesize large amounts of recombinant proteins but some of these molecules are not properly processed. Animal cells have to be used in some cases to obtain correct glycosylation, folding, etc. Milk of transgenic animals is considered as an excellent source of recombinant proteins. Mice are currently being used to evaluate the efficiency of gene constructs to be transferred in larger animals. It is generally considered that mice provide insufficient amount of milk to be the source of recombinant proteins. In the present paper it is shown that the isolated mammary gland from lactating mice isolated from their offsprings for one day released milk spontaneously when kept on ice for 2 to 18 hours. Essentially all the milk was released in this way. This procedure was applied to transgenic mice harboring the human growth hormone gene fused to the promoter of the rabbit whey acidic protein gene. The transgenic mice secreted the hormone at the concentration of 10 mg/ml (mean value). The hormone released with milk after incubation of the mammary gland on ice was undergraded and it kept its prolactin like activity. The method described in the present paper can therefore be used to prepare a few hundred milligrammes of recombinant proteins for biochemical or pharmaceutical studies. It may provide interesting information on the properties of recombinant proteins and thus be a first step towards the preparation of these molecules using farm animals.