Guy Kann

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.

The RU5 ('R') region from human leukaemia viruses (HTLV-1) contains an internal ribosome entry site (IRES)-like sequence.

RNA fragments containing the complete R region and the beginning of the U5 region (‘R’) from the human T cell leukaemia virus 1 (HTLV‐1) stimulated the translation of the second cistrons in bicistronic mRNAs. The 5′ untranslated region from SV40 early genes (SU) which was unable to stimulate translation of second cistrons amplified markedly the internal ribosome entry site (IRES) effect of the HTLV‐1 ‘R’ fragments. The ‘R’ regions from HTLV‐1 have therefore properties similar to internal ribosome entry sites (IRES) originally found in picornavirus. The beginning of the U5 region from HTLV‐1 contains a polypyrimidine sequence which is known to play an essential role in the IRES activity in picornavirus. The same experiments carried out using the ‘R’ region from bovine leukaemia virus (BLV) showed that this sequence has at most a weak IRES effect. One retroviruses HTLV‐1 and perhaps others contain therefore an IRES activity. Interestingly, the combined SU ‘R’ sequence worked efficiently with different cistrons, different promoters and in all tested cell lines, whereas the poliovirus IRES was active in CHO cells but not in the mouse mammary cell line HC11. The SU ‘R’ sequence may therefore preferably be used to generate active bicistronic mRNAs.

Maternal Olfaction Differentially Modulates Oxytocin and Prolactin Release during Suckling in Goats

In postparturient goats, olfactory recognition of the young allows the establishment of a selective bond between the mother and her kids. Once this bond is formed, the mother rejects alien young that attempt to suckle. We tested whether the development of the maternal selective bond in goats modulates prolactin (PRL) and oxytocin (OT) release in response to suckling. On day 37 of lactation, serial blood samples were taken during nursing of the mothers own or alien kid(s) in 10 intact/selective goats and in 10 goats rendered anosmic/nonselective through prepartum peripheral ZnSO(4) irrigation. Spontaneous nursing behavior was also studied weekly from day 7 to 30 of lactation, at which time milk production was measured. Maternal selectivity had no effect on PRL release, in contrast to OT release, which was significantly affected by this factor. Intact mothers released OT only when nursing their own kids, but not with aliens, while anosmic/nonselective dams showed an increase in OT levels regardless of the identity of the kids. In addition to these effects on maternal selectivity, the amplitude of the response of both hormones was lower in anosmic mothers than in intact mothers. Finally, nursing behavior and milk production were not significantly affected by anosmia. We conclude that maternal selective behavior in goats, which relies on the individual olfactory signature of the kid, modulates the OT, but not the PRL, response to suckling. In addition, perception of the smell of the young appears to have a general facilitatory effect, independent of the kids identity, on the release of both hormones.

Influence of the blood concentration of prolactin on the length of the sensitive period for establishing maternal behavior in sheep at parturition.

Abstract The fading of postpartum maternal interest for the neonate (sensitive period) in ewes separated from their young at lambing is delayed when parturition is induced with 20 mg of estradiol benzoate (EB). An experiment was carried out to investigate the role of prolactin in this phenomenon. The sensitive period was studied in three groups of parturient ewes. In all groups lambs were removed at birth and reintroduced to their mothers 24 hr later. Maternal acceptance was tested at this time. In group 1 (dexamethasone D), ewes were induced to lamb with dexamethasone (15 mg im). In group 2 (EB), ewes were treated with 20 mg of estradiol benzoate (im). In group 3 (EB + CB 154) ewes received 20 mg of EB as in group 2 and 1 mg of CB 154 (sc) every 12 hr to prevent the enhanced secretion of prolactin which normally occurs after EB injection. The concentration of prolactin was highest in group 2 (EB), lowest in group 3 (EB + CB), and intermediate in group 1 (D) (p ⩽ 0.001 between groups). By contrast, the proportion of ewes showing maternal behavior was similar in groups 2 and 3 ( 15 23 and 17 22 ), both of which differed from group 1 ( 3 22 ; p ⩽ 0.005 ). It is concluded that the lengthening of the sensitive period for establishing maternal behavior in sheep following EB induced parturition is not related with high levels of prolactin in the peripheral circulation.

The secretion of prolactin in intact and lutectomized pregnant ewes. Effect of the anti-progesterone steroid RU 486

To study the role, if any, of luteal factors in the control of prolactin secretion during the last two thirds of pregnancy in the ewe, we examined: a) the effect of RU 486 administration on prolactin secretion on days 97, 112 and 131 of pregnancy in five intact ewes and in five ewes from which the corpus luteum (CL) was removed on day 78 of pregnancy; and b) the secretory patterns of prolactin on days 60, 80, 100 and 120 of pregnancy in five intact ewes and in five ewes from which the CL was removed on day 70 of pregnancy. In a pilot experiment, we showed that daily i.v. injections (from day 91 to day 105 of pregnancy) of RU 486 at a dose of 50 mg caused a marked release of prolactin, without any effect on the secretion of progesterone and progression of pregnancy. In experiment 1, a single i.v. injection of 50 mg of RU 486 resulted in a significant (P < 0.01) increase in plasma prolactin concentrations on any day of pregnancy examined in the intact and lutectomized ewes. The prolactin responses (the maximum concentrations, the time to maximum concentrations and the area under the response curves) were not different between the two groups in any stage of pregnancy examined. In the two groups, spontaneous parturition occurred at term with alive lambs. There was no difference between the two groups in gestation length and lamb birth weight. In experiment 2, we showed that plasma concentrations of prolactin fluctuated in a pulsatile manner during the last two-thirds of pregnancy. The mean prolactin concentrations, the frequency and the amplitude of prolactin pulses were not significantly different between the intact and the lutectomized ewes in any stage of pregnancy examined. In conclusion, these experiments demonstrated that the ovine CL of pregnancy is not involved in the control of prolactin secretion in the ewe. The stimulation of prolactin secretion by the RU 486 is probably due to its anti-progesterone action exerted at the level of the receptor. The placental progesterone plays a central role in the control of prolactin secretion during the last two-thirds of pregnancy.