Mosaic expression of LacZ reporter gene controlled by 5′-regulatory sequences of alpha-S 1 -Casein gene in transgenic mice

The phenomenon of mosaic expression at the cellular level is frequently observed in tissues and organs of transgenic animals. The report concerns mosaicism in the progeny of five transgenic mouse founders carrying the LacZ reporter gene under the control of 5′-regulatory sequences of bovine and goat alpha-S ₁ -casein genes of various sizes. Cells positive for β-galactosidase E. coli activity were detected in lactating mammary glands of all transgenic females; however, the distribution of positive cells within the mammary glands was variable. We observed two types of mosaics, i.e., lobular (clonal) variegation when most or all lobular cells were positive for β-galactosidase and stochastic mosaicism when only single β-galactosidase positive cells were scattered within mammary glands. It is suggested that the stochastic type of mosaicism is realized in cells at the terminal stage of the differentiation of lactating glands, whereas the lobular one is developed from proliferating precursors capable of forming a whole lobule. The ectopic expression of the reporter gene was detected in the mandibular salivary gland in the offspring of two of the five founders No16 and No37, as well as in ovary follicles at the atrezia stage in the progeny of one of these founders. The low level of ectopic expression means that the 5′-flanked regulatory sequences of alpha-S ₁ -casein gene of various lengths used in the constructs ensure the reliable tissue-specific expression of the reporter gene.     

The effect of regulatory sequences of αSl-casein gene on the expression of the <Emphasis Type="Italic">lacZ</Emphasis>-gene in loach <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> L. transgenic embryos

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine αSl-casein gene tissue-specific promoter-enhancer region or highly homologous goat αSl-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1–3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the αSl-casein gene of goat was higher than with the promoter-enhancer region of the bovine αSl-casein gene. Thus, regulatory sequences of the bovine or goat αSl-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.     

Expression of the human granulocyte–macrophage colony stimulating factor (hGM-CSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice

Expression of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected “protective or enhancer effect” from the MAR element on the hGM-CSF gene expression was not observed.     

A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

Study on the Transfer of Foreign Genes into the Mussel Mytilus galloprovincialisLam. Eggs by Spermatozoa

The possibility of transferring exogenous DNA into eggs by mussel Mytilus galloprovincialisLam. sperms both with the use of certain methods of transfection and without them was studied. The efficacy of egg fertilization by sperms treated with foreign DNA and the development of larvae at early stages of embryogenesis were evaluated. Negative effects of the contact between mussel sperms and exogenous DNA on fertilization and subsequent development were noted. The proportion of developing larvae decreased with increasing DNA concentration and sperm exposure. Transfer of plasmids pCMVlacZ and pMTbGHinto eggs was observed in group crosses. With the use of PCR, foreign DNA sequences were found in the larvae at the stage of veliger 48 h after fertilization. An intense signal was recorded after sperm electroporation in 10% DMSO.     

Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha)

Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.     

Secretion of Biologically Active Human Granulocyte Colony-Stimulating Factor (G-CSF) in Milk of Transgenic Mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5′ and 3′ flanking promoter sequences of bovine alpha-S₁-casein gene (CSN1S1). Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3′ flanking region of bovine gene CSN1S1, but differed in size of the 5′ flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 μg/ml to over 1 mg/ml, in mice with construct pGCm1 and was low (up to 60 μg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCm1 and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF. __________ Translated from Genetika, Vol. 41, No. 10, 2005, pp. 1331–1337. Original Russian Text Copyright ? 2005 by Dvoryanchikov, Serova, Andreeva, Dias, Azevedo, Serov.