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.     

A Study of a Protein of the Annexin Group in Early Fish Development. II. Determination of Complete Amino Acid Sequence

We studied mRNA structure of 31 kDa annexin of zebrafish Brachydanio rerio using previously obtained 3"-terminal incomplete cDNA. The size of this protein mRNA was determined by Northern hybridization. PCR screening of cDNA library of zebrafish gastrula allowed us to obtain cDNA of the 5"-terminal regions of the mRNA. The primary structure of the protein deduced from the mRNA sequence allowed us to identify it as an annexin IV with threonine in position 6—a phosphorylation target for protein kinase C.     

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.     

Transient expression and activity of human DNA polymerase iota in loach embryos

Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.     

Influence of Regulatory Genes of Type 1 Human Immunodeficiency Virus on Proliferation and Differentiation of Murine Embryonic Stem Cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and green protein gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

Introduction of Human Recombinant Angiogenin at the Early Stages of Postnatal Development Inhibits the Growth of Mice

We studied the influence of recombinant DNA containing the cloned angiogenin gene, plasmid DNA without angiogenin gene, and purified recombinant angiogenin injected to Tg8 mice at the age of two days on the body mass of 28- and 40-day old mice. The body mass of mice that were injected with the cloned angiogenin gene or purified angiogenin was less than in the control mice. The body mice of Tg8 mice injected with recombinant DNA containing the cloned angiogenin gene did not increase from day 28 to day 40, while in the mice with purified recombinant angiogenin and control mice it increased by 24 and 57%, respectively. These data suggest that the elevated level of angiogenin at the early developmental stages inhibits the increase of body mass. The effect we described is related, in al likelihood, to the known inhibitory effect of angiogenin on protein synthesis.     

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.