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1.
Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice 总被引:8,自引:0,他引:8
C. Bruce A. Whitelaw Alan L. Archibald Stephen Harris Margaret McClenaghan J. Paul Simons A. John Clark 《Transgenic research》1991,1(1):3-13
We are studying the tissue-specific expression of the sheep milk-whey protein gene, β-lactoglobulin. We have used sequences
derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology
in transgenic sheep as a means of protein production. In the present study, a series of β-lactoglobulin hybrid genes and β-lactoglobulin
minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there
is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate
proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance
the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5′ flanking sequences
of β-lactoglobulin fused to a genomic minigene encoding human α-antitrypsin (α1AT) was expressed much more efficiently than an α1AT-cDNA construct containing the same β-lactoglobulin segment. Similarly, the intact β-lactoglobulin gene was expressed more
efficiently than the corresponding intronless β-lactoglobulin minigene. This effect was not seen in transient expression expriments
in baby hamster kidney cells when β-lactoglobulin-α1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing,
since the inclusion of the first β-lactoglobulin intron into cDNA constructs encoding human α1AT or β-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements
within introns may interact with the upstream 5′ flanking sequences of β-lactoglobulin and enable the latter to function efficiently
in the mammary gland of transgenic mice. 相似文献
2.
Christine P. Piotte Airlie K. Hunter Craig J. Marshall Murray R. Grigor 《Journal of molecular evolution》1998,46(3):361-369
Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two
forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected
in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA
libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those
for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins,
whereas trichosurin has similarities to the major urinary proteins of rodents.
Received: 28 October 1996 / Accepted: 19 May 1997 相似文献
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The robust and tissue-specific activation of the human growth hormone (hGH) gene cluster in the pituitary and placenta constitutes an informative model for analysis of gene regulation. The five-gene hGH cluster is regulated by two partially overlapping sets of DNase I hypersensitive sites (HSs) that constitute the pituitary (HSI, II, III and V) and placental (HSIII, IV, and V) locus control regions (LCRs). The single placenta-specific LCR component, HSIV, is located at −30 kb to the cluster. Here we generate a series of hGH/BAC transgenes specifically modified to identify structural features of the hGH locus required for its appropriate placental expression. We find that placental specificity is dependent on the overall multigene configuration of the cluster whereas the distance between the cluster and its LCR impacts the level of placental expression. We further observe that a major function of the placental hGH LCR is to insulate the transgene locus from site-of-integration effects. This insulation activity is linked to placenta-specific occupancy of the chromatin architectural protein, CTCF, at HSIV. These data reveal a remarkable combination of structural configurations and regulatory determinants that must work in concert to insure robust and tightly controlled expression from a complex multigene locus. 相似文献
5.
Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region
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The five genes of the human growth hormone (hGH) cluster are expressed in either the pituitary or placenta. Activation of the cluster is dependent on a locus control region (LCR) comprising pituitary- specific (HSI,II, -15 kb), placenta-specific (HSIV, -30 kb) and shared (HSIII, -28 kb; HSV, -32 kb) DNase I hypersensitive sites. Gene activation in the pituitary is paralleled by acetylation of a 32 kb chromatin domain 5' to the cluster centered at HSI,II. In the present study we observed that acetylation of this region in placental chromatin was discretely limited to shared HSIII and HSV. Transgenic studies revealed placenta-specific activation of linked genes by a determinant (P-element) located 2 kb 5' to each of the four placentally expressed genes. A localized peak of histone acetylation was observed at these P-elements in placenta but not pituitary. These data support a model for bifunctional action of the hGH LCR in which separate positive determinants, HSI,II and the P-elements, activate their respective target genes by tissue-specific recruitment of distinctly regulated histone acetyl transferase activities. 相似文献
6.
Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA)
in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG)
with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection.
The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish
dual transgenic mice of La-tPA /G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression
in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The
co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern
blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal
mice. The ratio of dual transgenes was 46.1% in F1 generation, and offspring’s sex ratio was normal. Hence a dual transgenic
mouse model was established for the study of co-expression foreign proteins in mammary gland. 相似文献
7.
Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status. 总被引:4,自引:0,他引:4
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There are now many mammalian examples in which single cell assays of transgene activity have revealed variegated patterns of expression. We have previously reported that transgenes in which globin regulatory elements drive the lacZ reporter gene exhibit variegated expression patterns in mouse erythrocytes, with transgene activity detectable in only a sub-population of circulating erythroid cells. In order to elucidate the molecular mechanism responsible for variegated expression in this system, we have compared the chromatin structure and methylation status of the transgene locus in expressing and non-expressing populations of erythrocytes. We find that there is a difference in the chromatin conformation of the transgene locus between the two states. Relative to active transgenes, transgene loci which have been silenced exhibit a reduced sensitivity to general digestion by DNase I, as well as a failure to establish a transgene-specific DNase I hypersensitive site, suggesting that silenced transgenes are situated within less accessible chromatin structures. Surprisingly, the restrictive chromatin structure observed at silenced transgene loci did not correlate with increased methylation, with transgenes from both active and inactive loci appearing largely unmethylated following analysis with methylation-sensitive restriction enzymes and by sequencing PCR products derived from bisulphite-converted genomic DNA. 相似文献
8.
Genomic constructs comprising the ovine -lactoglobulin gene are expressed in a position-independent manner in the mammary gland of transgenic mice. In some lines however, constitutive low-level transgene expression was detected in all other tissues. This ectopic expression presumably represents a position-dependent phenomenon since it was observed in only a proportion (40%) of the lines generated. Different lines of BLG transgenic mice displayed similar temporal patterns of ectopic expression. This pattern differed from that of BLG in the mammary gland. These data imply that the DNA elements that direct position-independent expression of -lactoglobulin transgenes in the mammary gland do not have the ability to insulate them from position effects in other tissues. Furthermore, the relatively high frequency and constitutive nature of ectopic expression suggests that transgene integration may not be totally random. 相似文献
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A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region. 总被引:12,自引:1,他引:11
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J Ellis K C Tan-Un A Harper D Michalovich N Yannoutsos S Philipsen F Grosveld 《The EMBO journal》1996,15(3):562-568
Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes. 相似文献
11.
Langley Brett Vilotte Jean-Luc Stinnakre Marie-Georges Whitelaw C. Bruce A. L'Huillier Phil J. 《Transgenic research》1998,7(3):205-212
In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine -lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus 相似文献
12.
E. Svetlova N. Avril-Fournout G. Ira P. Deschavanne J. Filipski 《Molecular & general genetics : MGG》1998,257(3):292-298
We have mapped the DNase I-hypersensitive sites (HSs) in Yeast Artificial Chromosomes (YACs) containing segments of human
chromosomal DNA. One of the five HSs found in a YAC carrying the β-globin gene cluster has been localised in the region, termed
HS2, that is DNase I hypersensitive in most human cells. We have also identified a class of HSs in YACs containing DNA from
the q11.2 band of human chromosome 21, which are located close to, or within, segments of the chromosome that are sensitive
to restriction enzymes recognizing CGCG tetranucleotides.
Received: 18 June 1997 / Accepted: 10 August 1997 相似文献
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14.
Transgenic mice containing a swine class I major histocompatibility complex (MHC) gene,PD1, express swine MHC (SLA) antigen. The tissue distribution of PD1 RNA parallels that observed in the swine, indicating that
the expression ofPD1 is regulated and thattrans-acting factors involved in this regulation have been conserved between the species. Although PD1 RNA levels were much greater
in transgenic spleen than in thymus, no difference in the chromatin organization of thePD1 gene was detected. In both tissues, a single DNase I hypersensitive site mapped within the 5′ flanking region. In vivo treatment
of the transgenics with mouse α, β-interferon increases PD1 expression in a number of tissues. In the spleen, this increase
parallels that observed for the endogenous transplantation antigen, Kb, but differs markedly from the differentiation antigen, Qa-2. Increases in cell surface expression of both PD1 and Kb occurred equally in splenic T- and B-cell populations following α,β-interferon treatment. In contrast, Qa-2 expression in
B cells was enhanced by α,β-interferon, whereas it was unaffected in T cells and thymocytes. 相似文献
15.
Binding of nogalamycin and adriamycin with Sarcoma-180 ascites tumor cell chromatin was studied by a spectrofluorometric method. There was significant reduction in the number of available drug binding sites per nucleotide when the chromatin was digested with DNase I for a period which releases only 7% of the chromosomal DNA. Results indicate preferential binding of these drugs with DNase I hypersensitive sites of chromatin. The DNase-I hypersensitive sites of chromatin were shown to correlate to the sequences required for gene expression. Further digestion with DNase I increases availability of drug binding sites, probably due to relaxation of the compact chromatin. 相似文献
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The local chromatin structure of the Shrunken-1 (Sh) gene of maize was probed by analyzing DNase I hypersensitivity. Sh encodes the gene for sucrose synthetase, a major starch biosynthetic enzyme, which is maximally expressed in the endosperm during seed maturation. In addition to general DNase I sensitivity, specific DNase I hypersensitive sites were identified in endosperm chromatin that mapped near the 5 end of the Sh gene. The pattern of hypersensitive sites and their relative sensitivity were altered in other non-dormant tissues that produce little or no enzyme. However, some changes in chromatin structure appear to be independent of Sh gene expression and may reflect general alterations associated with plant development. The chromatin structure of several sh mutations, induced by Ds controlling element insertions, was also analyzed. Although the insertions perturbed expression of the gene, there were no notable effects on local chromatin structure. 相似文献
19.
The sodium/iodide symporter (SLC5A5, also known as NIS) is a transmembrane glycoprotein. Physiologically, iodide transportation in the mammary gland occurs during late pregnancy
and lactation. To identify factors that may regulate this process at different iodine levels, we have studied the expression
of NIS gene and protein in cultured mammary gland explants from lactating mice by real-time quantitative PCR and In-Cell Western
methods. Mammary gland cells were grown in media with different levels of iodine for 24 h. The iodine treatment groups consist
of low iodine group I (LI-I, 0 μg/l), low iodine group II (LI-II, 5 μg/l), control group (C, 50 μg/l), high iodine group I
(HI-I, 3,000 μg/l), and high iodine group II (HI-II, 10,000 μg/l). The cells were then incubated with or without insulin-like
growth factor I (IGF-I) or transforming growth factor β1 (TGF-β1) for another 24 h. We found that iodine inhibited NIS mRNA and protein expression in a dose-dependent manner. IGF-I and TGF-β1 further decreased NIS mRNA and protein expression that iodine inhibited at different iodine levels. In summary, we have shown that iodine downregulated
NIS expression in cultured mammary gland explants from the lactating mouse. IGF-I and TGF-β1 inhibited NIS mRNA and protein expression in the mammary gland under different iodine levels. 相似文献
20.
Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture
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Fang X Sun J Xiang P Yu M Navas PA Peterson KR Stamatoyannopoulos G Li Q 《Molecular and cellular biology》2005,25(16):7033-7041
Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions. 相似文献