Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development.

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis-regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.

Characterisation of two identical independent non-homologous integration sites in mouse embryonic stem cells.

On analysis of 46 Geneticin-resistant (GtR) cell lines, derived by electroporation of mouse embryonic stem (ES) cells with a promoterless neo vector, we observed that in two independently derived cell lines, the vector had integrated into the same locus. The sequence flanking the vector integration site in both cell lines was cloned and sequenced. The vector had integrated into a 3 to 6-bp region in both cell lines. No homology is observed between the integration site sequence and the vector sequence.

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.

Differential extra-renal expression of the mouse renin genes.

We have used RNase-protection analyses to study renin gene expression in one- and two-gene mouse strains. The RNase-protection assay is capable of discriminating between the transcripts from the different renin genes. In a two-gene strain containing Ren-1D and Ren-2, we demonstrate transcriptional activity from Ren-1D in kidney, submandibular gland (SMG), testes, liver, brain and heart. Ren-2 is clearly expressed in kidney, SMG and testes. Similar analyses of one gene strains (containing Ren-1C only) show expression in kidney, SMG, testes, brain and heart. We cannot detect renin mRNA in the liver of these mice. Ren-1C and Ren-1D thus display quite different tissue-specificities. In order to determine whether the different tissue-specificities of the highly homologous Ren-1C and Ren-1D genes are due to different trans-acting factors in the different mouse strains or to different cis-acting DNA elements inherent to the genes, we introduced a Ren-1D transgene (Ren-1*) into a background strain containing only the Ren-1C gene. The transgene exhibits the same tissue-specificity as the Ren-1D gene of two-gene strains suggesting the presence of different cis-acting DNA elements in Ren-1C and Ren-1D.

Widespread expression of human alpha 1-antitrypsin in transgenic mice revealed by in situ hybridization.

In situ hybridization is a powerful means of identifying sites of gene expression. We used this technique to examine the spatial and developmental control of transcription of the human alpha 1-antitrypsin (alpha 1 AT) gene in transgenic mice carrying this gene and extensive 5'- and 3'-flanking sequences. In addition to expression in yolk sac and liver, human alpha 1AT RNA was detected in gut, stomach, pancreas, nasal epithelium, pharynx, bronchi, spinal ganglia, and ossifying cartilage of transgenic fetuses at 14.5 days post coitum (dpc). In transgenic adults, expression was no longer found in the pancreas but was found in the kidney and salivary gland. In each tissue, expression was confined to a specific cell population. This pattern of alpha 1AT expression was found to correlate with that seen in several fetal and adult human tissues. These results suggest a wider role of alpha 1AT in human physiology and development than previously suspected, and they demonstrated the potential value of this approach in delineating the physiological role of human proteins. Expression of the endogenous alpha 1AT gene in mice was confined to a limited, but overlapping, set of tissues, suggesting that the cis-acting DNA sequences that regulate the expression of the human and mouse genes interact differently with transcription factors present in mouse cells.

Two maternally derived X chromosomes contribute to parthenogenetic inviability.

In certain extraembryonic tissues of normal female mouse conceptuses, X-chromosome-dosage compensation is achieved by preferential inactivation of the paternally derived X. Diploid parthenogenones have two maternally derived X chromosomes, hence this mechanism cannot operate. To examine whether this contributes to the inviability of parthenogenones, XO and XX parthenogenetic eggs were constructed by pronuclear transplantation and their development assessed after transfer to pseudopregnant recipients. In one series of experiments, the frequency of postimplantation development of XO parthenogenones was much higher than that of their XX counterparts. This result is consistent with the possibility that two maternally derived X chromosomes can contribute to parthenogenetic inviability at or very soon after implantation. However, both XO and XX parthenogenones showed similar developmental abnormalities at the postimplantation stage, demonstrating that parthenogenetic inviability is ultimately determined by the possession of two sets of maternally derived autosomes.

Tissue-specific expression of the human type II collagen gene in mice.

Type II collagen is crucial to the development of form in vertebrates as it is the major protein of cartilage. To study the factors regulating its expression we introduced a cosmid containing the human type II collagen gene, including 4.5 kilobases of 5' and 2.2 kilobases of 3' flanking DNA, into embryonic stem cells in vitro. The transformed cells contribute to all tissues in chimeric mice allowing the expression of the exogenous gene to be studied in vivo. Human type II collagen mRNA is restricted to tissues showing transcription from the endogenous gene and human type II collagen is found in extracellular matrix surrounding chondrocytes in cartilage. The results indicate that the cis-acting requirements for correct temporal and spatial regulation of the gene are contained within the introduced DNA.

Species- and tissue-specific expression of human alpha 1-antitrypsin in transgenic mice.

alpha 1-Antitrypsin (alpha 1AT) is an abundant serum protein whose major site of synthesis is in the hepatocyte. alpha 1AT transcripts are also present, albeit at a lower level, in a variety of other human tissues. This pattern of expression is partly related to initiation of transcription at sites with distinct tissue specificities. The mouse alpha 1AT gene, in contrast, is more strictly liver specific in its expression. To explore the regulation of the alpha 1AT gene we have microinjected a cosmid insert carrying the human gene into fertilized mouse eggs. In three lines obtained from transgenic mice, inheritance of copies of the human gene is accompanied by a high serum concentration of the human protein. Human alpha 1AT RNA accumulates to the highest level in liver of transgenic animals. The presence of transcripts in other tissues indicates that the human pattern of expression is maintained, whereas the temporal activity of the introduced gene parallels that of the endogenous one during mouse embryogenesis.

The development of XO gynogenetic mouse embryos.

Diploid gynogenetic embryos, which have two sets of maternal and no paternal chromosomes, die at or soon after implantation. Since normal female embryos preferentially inactivate the paternally derived X chromosome in certain extraembryonic membranes, the inviability of diploid gynogenetic embryos might be due to difficulties in achieving an equivalent inactivation of one of their two maternally derived X chromosomes. In order to investigate this possibility, we constructed XO gynogenetic embryos by nuclear transplantation at the 1-cell stage. These XO gynogenones showed the same mortality around the time of implantation as did their XX gynogenetic counterparts. This shows that the lack of a paternally derived autosome set is sufficient to cause gynogenetic inviability at this stage. Autosomal imprinting and its possible relation to X-chromosome imprinting is discussed.

Protein synthetic patterns of tissues in the early chick embryo.

Tissues dissected from early chick embryos were labelled in vitro with [35S]methionine, and their patterns of polypeptide synthesis investigated using the technique of two-dimensional (2-D) polyacrylamide gel electrophoresis. Apart from providing a preliminary description of the molecular changes associated with the processes of gastrulation and segmentation in the chick embryo, this study has revealed a number of polypeptides that may be useful as markers of cell type or function. The protein synthetic patterns of hypoblast from early and late gastrulae (stages 2 and 4, respectively: Hamburger & Hamilton, 1951) and of definitive endoblast and junctional endoblast from late gastrulae all resemble one another closely, but differ markedly from that of the epiblast at either stage. The lower layer tissues are characterized by the presence of eleven polypeptides that are largely absent from the epiblast. These findings are discussed with reference to current theories on the origins of the lower layer tissues. Comparisons between the 2-D patterns for tissues dissected from gastrulae and from embryos undergoing segmentation (stage 12) have revealed ten polypeptides showing stage-specific rather than tissue-specific expression. Apart from these ten polypeptides, the 2-D patterns for epiblast and ectoderm were practically identical, and distinguishable from those of other tissues by a lack of any unique polypeptides. On the other hand, stage-4 endoblast and stage-12 endoderm differed in the expression of many polypeptides. One polypeptide was found that may be considered as a marker of mesodermal cell type, as it was present in lateral plate, segmental plate and somitic mesoderm, but not in tissues of the other germ layers. Lateral plate could be distinguished from the other mesodermal tissues in the expression of a number of polypeptides, but the similarity in the 2-D patterns for segmental plate and somites suggest that the separation of somites from the anterior end of the segmental plate is not accompanied by the synthesis of new polypeptides.

Inviability of parthenogenones is determined by pronuclei, not egg cytoplasm.

Parthenogenetic mouse embryos pose an interesting problem in the study of early mammalian development. Haploid or diploid parthenogenones, resulting from spontaneous or experimental activation of unfertilized eggs, will undergo apparently normal preimplantation development but die in the early post-implantation stages. However, in aggregation chimaeras with fertilized embryos, parthenogenetic embryos have the ability to differentiate into many tissue types, including gametes which can give rise to normal offspring. Furthermore, it has been reported that viable young were obtained from the transfer of inner cell-mass nuclei of parthenogenetic blastocysts to enucleated fertilized eggs. These observations suggest that sperm have some additional role, apart from restoring a complete genome, that is necessary for normal development. To investigate whether sperm-related modifications to the egg cytoplasm are important, we have used an efficient nuclear transfer technique in which a complete karyoplast, comprised of pronuclei, surrounding cytoplasm and a portion of the egg plasma membrane, is transferred utilizing Sendai virus membrane fusion. Embryos produced by the transfer of pronuclei from diploid parthenogenetic eggs to enucleated fertilized eggs died very soon after implantation, whereas viable young were obtained from the transfer of fertilized egg pronuclei into enucleated parthenogenetic eggs. This shows that the death of parthenogenones is not due to a lack of cytoplasmic factors from the sperm.

Changes in protein synthesis during differentiation of embryonal carcinoma cells, and a comparison with embryo cells.

Two-dimensional electrophoresis was used to find changes in protein synthesis occurring as pluripotent embryonal carcinoma (EC) cells differentiate to give embryoid bodies in vitro. 2-D patterns from other embryonic cell lines, and from the inner cell mass (ICM) cells of mouse embryos, were also analysed for the expression of those proteins showing some change during embryoid body formation and for overall differences between these and the EC cells. Most changes in protein synthesis occurred before 12 h but endoderm was not discerned morphologically on the outside of EC cell clumps until at least 18 h after their suspension. The number of changes occurring is small compared with the number of polypeptides resolved, but is in line with similar studies. Comparisons with nullipotent EC cells and an endodermal cell line have allowed these changes to be assigned, tentatively, to the different cell types within embryoid bodies, and may allow them to be used as markers of differentiation. Comparisons between the 2-D patterns derived from ICMs and EC cells reveal substantial differences between the two that might not have been expected from their developmental homology. The importance of these differences to their pluripotentiality is discussed.