The implementation of mental health care reform in Israel.
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Community Mental Health Services↗
Biomedical subjects
M Shani
Publications and source records attributed to M Shani.
Ectopic expression of beta-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation and in situ localization.
We produced transgenic mice carrying the native sheep beta-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains. In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.
Animals↗
Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice.
A new series of expression vectors, each comprised of the beta-lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSA in vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levels in vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml-1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml-1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trend in vitro with the trend of expression in vivo (% which express) at detectable levels (p = 0.0015) and at the level of greater than 0.1 mg ml-1 (p = 0.0156) was demonstrated. A weak correlation existed (p = 0.0526) at in vivo levels of 1 mg ml-1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.
Animals↗
Testicular amplification and impaired transmission of human butyrylcholinesterase cDNA in transgenic mice.
Gene amplification occurs frequently in tumour tissues yet is, in general, non-inheritable. To study the molecular mechanisms conferring this restraint, we created transgenic mice carrying a human butyrylcholinesterase (BCHE) coding sequence, previously found to be amplified in a father and son. Blot hybridization of tail DNA samples revealed somatic transgene amplifications with variable restriction patterns and intensities, suggesting the occurrence of independent amplification events, in 31% (11/35) of mice from the FII generation but in only 3.5% (2/58) of the FIII and FIV generations. In contrast, > 10-fold amplifications of the BCHE transgene and the endogenous acetylcholinesterase and c-raf genes appeared in both testis and epididymis DNA from > 80% of FIII mice. Drastic, selective reductions in testis BCHEmRNA but not in actin mRNA were detected by the PCR amplification of testis cDNA from the transgenic mice, and apparently resulted in the limited transmission of amplified genes. The testicular amplification of the BCHE transgene may potentially represent a general phenomenon with clinical implications in human infertility.
Animals↗
Health in Gaza.
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Delivery of Health Care↗
[Clinical guidelines. Background, methodology and significance].
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Humans↗
Ectopic expression of MyoD1 in mice causes prenatal lethalities.
A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether MyoD1 is a dominant muscle regulator in vivo, mouse fertilized eggs were microinjected with a beta-actin/MyoD1 gene. Ectopic expression of MyoD1 during mouse embryogenesis led to embryonic lethalities, the cause of which is not known. Transgenic embryos died before midgestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control littermates, differentiated normally into tissues representative of all three germ layers. In most transgenic embryos there was no indication of myogenic conversion. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. Forced expression of MyoD1 was associated with the activation of myogenin and MLC2 (but not myf5 or MRF4) genes in non-muscle cell types, demonstrating the dominant regulatory function of MyoD1 during development. These results demonstrate that ectopic MyoD1 expression and activation of myogenin and MLC2 have no significant effects in the determination of cell lineages or the developmental fate of differentiated mesodermal and ectodermal cell lineages.
Actins↗
Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice.
Transgenic mice were produced, carrying hybrid genes comprised of the ovine beta-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences. In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2- to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.
Animals↗
The expression of the regulatory myosin light chain 2 gene during mouse embryogenesis.
The fast skeletal muscle myosin light chain 2 (MLC2) gene is expressed specifically in skeletal muscles of newborn and adult mice, and has no detectable sequence homology with any of the other MLC genes including the slow cardiac MLC2 gene. The expression of the fast skeletal muscle MLC2 gene during early mouse embryogenesis was studied by in situ hybridization. Serial sections of embryos from 8.5 to 12.5 days post coitum (d.p.c.) were hybridized to MLC2 cRNA and to probes for the myogenic regulatory genes MyoD1 and myogenin. The results revealed different temporal and spatial patterns of hybridization for different muscle groups. MLC2 transcripts were first detected 9.5 d.p.c. in the myotomal regions of rostral somites, already expressing myogenin. Surprisingly, at the same stage, a weak MLC2 signal was also detected in the cardiomyocytes. The cardiac expression was transient and could not be detected at later stages while the myotomal signal persisted and spread to the more caudal somites, very similar to the expression of myogenin. Beginning from 10.5 d.p.c., several extramyotomal premuscle cells masses have been demarcated by MyoD1 expression. MLC2 transcripts were detected in only one of these cell masses. Although, transcripts of myogenin were detected in all these cell masses, the number of expressing cells was significantly lower than that observed for MyoD1. By 11.5 d.p.c., all three hybridization signals colocalized in most extramyotomal muscle-forming regions, with the exception of the diaphragm and the hindlimb buds, where only few cells expressed MLC2 and more cells expressed MyoD1 than myogenin. At 12.5 d.p.c., all three studied genes displayed a similar spatial pattern of expression in most muscle-forming regions. However, in some muscles, the MyoD1 signal spread over more cells compared to myogenin or MLC2. Our results are consistent with the suggestion that multiple myogenic programs exist for myoblasts differentiating in the myotome and extramyotomal regions.
Animals↗
Bovine dinucleotide repeat polymorphism at the ARO26 locus.
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Alleles↗
Bovine dinucleotide repeat polymorphism at the ARO28 locus.
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Alleles↗
Regulatory elements that control the lineage-specific expression of myoD.
The molecular basis of skeletal muscle lineage determination was investigated by analyzing DNA control elements that regulate the myogenic determination gene myoD. A distal enhancer was identified that positively regulates expression of the human myoD gene. The myoD enhancer and promoter were active in myogenic and several nonmyogenic cell lines. In transgenic mouse embryos, however, the myoD enhancer and promoter together directed expression of a lacZ transgene specifically to the skeletal muscle lineage. These data suggest that during development myoD is regulated by mechanisms that restrict accessibility of myoD control elements to positive trans-acting factors.
Animals↗
Expression of human serum albumin in the milk of transgenic mice.
We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep beta-lactoglobulin (BLG) 5'-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5'-sequences demonstrated that 3 kb of 5'-sequences were sufficient to support high levels of expression of BLG, and that the longer 5'-sequences did not improve upon the levels of expression. As such, the 3 kb 5'-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml-1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 micrograms ml-1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.
Animals↗
Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.
The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.
Aging↗
The consequences of a constitutive expression of MyoD1 in ES cells and mouse embryos.
A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones tested, carrying single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 genes but not the endogenous MyoD1, MRF4, myf5, skeletal muscle actin or myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers was observed only when the transfected cells were allowed to differentiate, via embryoid bodies, in low mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and myf5 genes and in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of MyoD1 was not required for myogenesis. Interestingly, no preferential myogenesis was observed when the transfected ES cells were allowed to differentiate in vivo to teratocarcinomas. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function. Second, MyoD1 function in ES cells, even under environmental conditions that favour differentiation, is not dominant (incomplete penetrance). Third, that the exogenous MyoD1 transactivates the endogenous myogenin and MLC2 genes in ES cells. No live transgenic mice could be produced following microinjection of the beta-actin/MyoD1 gene into the pronuclei of fertilized eggs. Transgenic embryos died before mid gestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control litermates, differentiated into tissues representative of all three germ layers. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. These results demonstrate again that MyoD1 is not a dominant regulatory factor.
Actins↗
Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.
A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.
Animals↗
Demethylation of CpG islands in embryonic cells.
DNA in differentiated somatic cells has a fixed pattern of methylation, which is faithfully copied after replication. By contrast, the methylation patterns of many tissue-specific and some housekeeping genes are altered during normal development. This modification of DNA methylation in the embryo has also been observed in transgenic mice and in transfection experiments. Here we report the fate in mice of an in vitro-methylated adenine phosphoribosyltransferase transgene. The entire 5' CpG island region became demethylated, whereas the 3' end of the gene remained modified and was even methylated de novo at additional sites. Transfection experiments in vitro show that the demethylation is rapid, is specific for embryonic cell-types and affects a variety of different CpG island sequences. This suggests that gene sequences can be recognized in the early embryo and imprinted with the correct methylation pattern through a combination of demethylation and de novo methylation.
Adenine Phosphoribosyltransferase↗
[Pitfalls in international comparisons of health data and indices].
Comparison of published data and health indices from different countries with different health systems is subject to many pitfalls. Comparison of national expenditure for health care based on purchasing power of the currency may be misleading if the purchasing power of the health services is ignored. Comparisons may also be misleading if they ignore national geographic and demographic structures. Government and health authorities often quote different sets of data and use different terminology and definitions. This article stresses the disparity in the definition of medical manpower and points out differences relating to ratios of manpower to population and to per capita spending. Also addressed is the importance of the qualitative and value aspects of health systems not usually involved in comparison of international health indices. In conclusion, safety measures and precautions such as choosing the right index for the right purpose, adjustment of indices to the purchasing power parity of health, demographics, etc., should be used when conducting health care analyses.
Data Collection↗