Transgenic mice with I-A on islet cells are normoglycemic but immunologically intolerant.

Insulin-dependent diabetes mellitus (IDDM) is caused by a specific loss of the insulin-producing beta cells from pancreatic Langerhans islets. It has been proposed that aberrant expression of major histocompatibility complex (MHC) class II molecules on these cells could be a triggering factor for their autoimmune destruction. This proposal was tested in transgenic mice that express allogeneic or syngeneic class II molecules on the surface of islet cells at a level comparable with that normally found on resting B lymphocytes. These animals do not develop diabetes, nor is lymphocyte infiltration of the islets observed. This immunological inactivity does not result from tolerance to the "foreign" class II molecules.

B-cell control region at the 5' end of a major histocompatibility complex class II gene: sequences and factors.

Transcription of major histocompatibility complex class II genes is elaborately regulated. Mouse class II genes are transcribed primarily in B cells, peripheral macrophages and interdigitating cells, and thymic cortical and medullary cells. In this study, we began to identify the DNA sequences and protein factors that control expression of a class II gene in B cells, addressing in particular how closely they resemble those that regulate immunoglobulin gene expression. We describe a region upstream of the E alpha gene that is crucial for its transcription in the B cells of transgenic mice but is less important in cultured B-cell lines. The sequence of this region reveals several familiar motifs, including a second X-Y pair reminiscent of that residing in the promoter-proximal region of all class II genes, a B motif strikingly homologous to that associated with the immunoglobulin kappa gene enhancer, several Ephrussi motifs, and a Pu box-like sequence very similar to that implicated in simian virus 40 and lymphotrophic papovavirus expression in B cells. Careful study of the proteins that bind specifically to these different motifs prompts us to suggest that major histocompatibility complex class II and immunoglobulin genes rely on quite different factors to achieve B-cell-specific expression.

H-ras induced transformation of mammary epithelium is favoured by increased oncogene expression or by inhibition of mammary regression.

The promoter of the mammary specific murine whey acidic protein gene was used to direct Ha-ras expression in different lines of transgenic mice. We found that this promoter contains a tissue specific enhancer which directed expression in both orientations albeit to different levels. We used this feature to generate low and high ras expressing transgenic lines. The reversed orientation led to a weak expression in lines 3 and 58 and to a tumor frequency of 2%. In contrast, 72% of mice from line 25 showing high ras expression developed mammary tumors. Nulliparity is one risk factor for human breast cancer, suggesting a protective effect of post-lactational mammary regression. In order to investigate the effect of post-lactational regression, the low tumor frequency lines were crossed with mice expressing ubiquitously the human growth hormone gene, which induces permanent development of the mammary epithelium. Indeed, mammary tumors were observed in 76% of double transgenic females. Thus, the tumorigenic potential of the ras oncogene in mammary cells in vivo correlates with the level of its expression and with the developmental history of the mammary gland. Transformation coincides with the escape of oncogene expression from the regulation of the Wap promoter and the extinction of endogenous Wap gene expression.

Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk.

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.

Precocious mammary gland development and milk protein synthesis in transgenic mice ubiquitously expressing human growth hormone.

A chimeric gene comprising the hydroxymethylglutaryl coenzyme-A reductase promoter and the human GH (hGH) genomic sequences was used to create transgenic mice expressing hGH in all tissues. In transgenic females, morphological development of the mammary gland and milk protein (WAP) expression commences at 3 weeks of age. At 8 weeks of age the mammary gland is morphologically and functionally comparable to that normally reached after 14-15 days of gestation. Precocious development correlated with local expression of hGH in mammary gland. Organ culture in the presence of different lactogenic hormones revealed that insulin and hydrocortisone are sufficient to maintain transcription of the WAP gene in transgenic mammary gland. In contrast, WAP transcription in normal gland required either hGH or PRL in addition to insulin and hydrocortisone. However, the effect of hGH on mammary differentiation does not appear to be solely mediated through an interaction with PRL receptors, since PRL, when added to cultured mammary tissues, did not elicit an equivalent response.

Transvaginal sonographically controlled follicle puncture for oocyte retrieval.

Transvaginal sonographically guided follicle aspiration under local anesthesia was performed on more than 100 patients and compared favorably with the other techniques that have been proposed to retrieve oocytes for in vitro fertilization. Sonography employs a sector scanner placed on the abdomen, and the needle is introduced through the posterior fornix of the vagina into the cul-de-sac and the ovary. In case of a high ovary, a transvaginal-transvesical variation may be used. The method is not painful and is easy to learn and perform. The sole adverse incidents have involved inadvertent venous puncture with no sequelae. The number and quality of recovered oocytes are good, and five normal children conceived after this method of retrieval were recently born. The technique permits substantial simplification of egg recovery for in vitro fertilization, which can now be performed in an outpatient setting without the risk and expense of laparoscopy and general anesthesia or the discomfort of transabdominal-transvesical ultrasound-guided aspiration.

[Towards a single embryo transfer: predictive value of uterine and embryological parameters in assisted reproduction outcome]. / Vers un transfert mono-embryonnaire : prise en compte conjointe des paramètres embryonnaires et utérins en assistance médicale à la procréation.

OBJECTIVE: The transfer of a single embryo would avoid obstetrical and neonatal complications due to multiple pregnancies. We studied the clinical value of both uterine and embryological parameters to define precise conditions allowing a single embryo transfer without decreasing pregnancy rates. PATIENTS AND METHODS: Endometrial parameters expressed by a uterine score together with biological criteria of 131 in vitro fertilization or intracytoplasmic sperm injection attempts were retrospectively analysed. RESULTS: Two hundred and sixty-two day-3 embryos were replaced through 131 transfers. Fifty-seven pregnancies were induced and 16 twins were obtained. The clinical pregnancy rate was 35.9% and the embryonic implantation rate 24.0%. After the transfer of two embryos, successful implantation was determined by the occurrence of top-quality embryos and simultaneously by a receptive endometrium. The uterine pulsatility index was significantly decreased for twin compared to singleton pregnancies. DISCUSSION AND CONCLUSION: This study confirms that a uterine score constitutes a powerful tool for evaluating the uterine receptivity. This parameter has to be taken into account as well as the embryonic quality, in order to optimise the success rate. Young patients with at least two top-quality embryos available, a high uterine score and a low pulsatility index, have a high risk of multiple births and are suitable for a single-embryo transfer.

[Twin birth after preimplantation diagnosis for cystic fibrosis]. / Naissance gémellaire après diagnostic préimplantatoire pour mucoviscidose.

OBJECTIVE: Cystic fibrosis is a common autosomal recessive disease most often caused by a deletion (delta F508) in the CFTR gene. It is the most common indication for preimplantaion genetic diagnosis which allows genetic analysis of embryos obtained after in vitro fertilization and transfer of unaffected embryos into the patient's uterus. PATIENTS AND METHODS: We report the first preimplantation genetic diagnosis performed in Strasbourg for a couple at risk of having a child affected by severe cystic fibrosis due to a homozygous delta F508 mutation. Three days after fertilisation, embryos obtained after intra-cytoplasmic testiculare sperm injection were biopsied and analysed. PCR amplification of the genomic fragment containing the delta F508 locus allowed detection of the delta F508 mutation and transfer only of the unaffected embryos. RESULTS: Three embryos were transferred after this preimplantation genetic diagnosis. A twin pregnancy was obtained and the babies born from this cycle are both exempt from the mutation. CONCLUSIONS: Preimplantation genetic diagnosis for the cystic fibrosis delta F508 mutation is now available in our centre. In this report, we could resolve both the problem of infertility and the risk of transmission of a severe form of cystic fibrosis. Preimplantation genetic diagnosis is also available for other mutations involved in cystic fibrosis and also for other genetic diseases.

Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 2. The coding region (nucleotides 69-236).

In the preceding paper it was shown that the first A-U-G codon in tobacco mosaic virus RNA is separated from the 5' terminus by a sequence of 68 nucleotides devoid of internal guanosine residues. In this paper we present the sequence of 165 residues immediately following the first potential initiation codon. The characterized sequence contains four nonsense codons but none are in phase with the prospective initiation codon. Several lines of evidence, including direct characterization of the portion of the RNA molecule which binds to and is protected by the ribosome in the course of initiation, all support the idea that the A-U-G at position 69-71 is a functional initiation signal for viral protein synthesis.

Preparing for preimplantation genetic diagnosis in France.

Preimplantation genetic diagnosis (PGD) allows the detection of genetic defects before implantation, thus circumventing the possible need for abortion. France's current legislation allows the practice of PGD under certain circumstances which include the prerequisite agreement of the French health authority. Unfortunately, to enact the pending 'bioethical law', voted in July 1994, a decree still needs to be published. So, for the moment, although we know that PGD should be authorized, its practice is currently impossible in France. In order to prepare for licensing, we are setting up the relevant technologies, by performing biopsy on mouse embryos and fluorescent in-situ hybridization (FISH) experiments on human lymphoblast cells. We briefly describe the French legal situation with regard to PGD and the work we have performed in this context to obtain the licensing to offer PGD to patients. After a period of preparation, 95.9% of biopsies were successful and up to 95.4% of the biopsied blastomeres were properly spread onto slides. Biopsied and control mouse embryos were reimplanted into pseudopregnant females and similar birth rates were obtained (34.4 and 30.9% respectively). In these experiments we noticed a birth delay of 12-24 h for the biopsied embryos compared with the controls. Furthermore, scanning electron microscopy of the biopsied embryos allowed assessment of the hole made by the Tyrode's acid. By intercrossing adults derived from biopsied embryos for two successive generations, it was shown that the biopsy did not generate defects affecting their reproductive ability. FISH experiments were performed using specific probes for chromosomes X, Y and 1 on nuclei spread by a conventional protocol or a Tween/HCl blastomere spreading protocol; in the latter case, slides with 1-5 cells were prepared. A similar percentage of correct X,Y,1,1 signal was obtained from all three types of spreading, varying from 85.5 to 89.9%.

Correcting an immune-response deficiency by creating E alpha gene transgenic mice.

We have introduced a cloned Ek alpha gene into embryos of mice incapable of expressing their endogenous E alpha genes. The transcription of Ek alpha was accurate and tissue-specific in the resulting transgenic lines and, in macrophages, Ek alpha transcription could be induced by gamma-interferon. The injected gene conferred on the genetically deficient host strain the ability to mount an immune response to a synthetic polymer.