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1.
Expression of human serum albumin in the milk of transgenic mice 总被引:7,自引:0,他引:7
Moshe Shani Itamar Barash Margret Nathan George Ricca George H. Searfoss Itzhak Dekel Alexander Faerman David Givol David R. Hurwitz 《Transgenic research》1992,1(5):195-208
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 β-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 μg 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. 相似文献
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Itamar Barash Alexander Faerman Tamar Ratovitsky Raisa Puzis Margaret Nathan David R. Hurwitz Moshe Shani 《Transgenic research》1994,3(3):141-151
We produced transgenic mice carrying the native sheep -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. 相似文献
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Establishment and initial characterization of the ovine mammary epithelial cell line nish 总被引:4,自引:0,他引:4
Neta Ilan Itamar Barash Elisha Gootwine Moshe Shani 《In vitro cellular & developmental biology. Animal》1998,34(4):326-332
Summary Analysis of the molecular mechanisms involved in the differentiation and formation of the characteristic three-dimensional
structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program
of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived
spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete β-lactoglobulin (BLG) in an ECM (extracellular
matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells
cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously
needed to maintain BLG expression. Stable transfection of NISH cells with BLG/Human Serum Albumin (HSA) hybrid gene constructs
revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in cells transfected with
hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic
sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and organogenesis
of mammary epithelial cells under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for
the transgenic mouse model in evaluating the potential of gene constructs to be efficiently expressed in the mammary gland
of transgenic farm animals. 相似文献
6.
I. A. Serova L. E. Andreeva N. V. Khaidarova L. P. B. Dias G. A. Dvoryanchikov I. A. Burkov N. V. Baginskaya 《Cell and Tissue Biology》2009,3(5):409-416
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. 相似文献
7.
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
8.
《Gene》1997,193(2):239-243
We have analysed the expression of β-lactoglobulin (BLG) gene constructs with combinations of introns deleted to further define the role of intronic regions in directing position-independent mammary expression of BLG transgenes. Intron removal had no obvious effect on hormonal induction of BLG expression in vitro but dramatically reduced expression in vivo, in that removal of intron pairs always resulted in a proportion of the transgenic lines generated failing to express the transgene in the mammary gland. Position-dependent expression was seen for all intron-deleted transgenes regardless of which introns were removed and the ability of the intron-deleted transgenes to be expressed bore no relationship to transgene copy number. Thus, intron removal per se increases the sensitivity of BLG transgenes to position effects. 相似文献
9.
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
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Genomic constructs comprising the ovine β-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 β-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
14.
Chrenek P Ryban L Vetr H Makarevich AV Uhrin P Paleyanda RK Binder BR 《Transgenic research》2007,16(3):353-361
Transgenic founder rabbits carrying a gene construct consisting of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb
of the human clotting factor VIII (hFVIII) cDNA and 4.6 kb of 3′ flanking sequences of mWAP gene were crossed for three generations.
All transgenic animals showed stable transgene transmission. Transgenic females showed high level of recombinant hFVIII (rhFVIII)
mRNA expression in biopsed mammary gland tissues, while marginal expression of rhFVIII mRNA was observed in the spleen, lung
and brain. No adverse effects of ectopic expression on the physiology of the rabbits were observed. Expression was not detected
in the liver, kidney, heart and skeletal muscle. In transgenic females derived from three generations, rhFVIII protein was
secreted from the mammary gland of lactating females, as shown by Western blotting. Biological activity of rhFVIII protein,
as revealed in clotting assays was ranged from 0.012 to 0.599 IU/ml corresponding to 1.2% and 59.9% of the hFVIII level in
normal human plasma. No apparent effect of secreted rhFVIII on the milk performance of rabbits was observed. Our results confirm
the possibility of producing a significant amount of a biologically active rhFVIII in the mammary gland of established transgenic
rabbit lines. 相似文献
15.
Chromosomal sequences flanking an efficiently expressed transgene dramatically enhance its expression 总被引:3,自引:0,他引:3
The expression of transgenes in mice is influenced strongly by their site of integration in the genome. To test whether the chromosomal sequences immediately flanking a site of integration could positively influence expression we isolated the 5' and 3' chromosomal sequences from an efficiently expressed transgenic locus. These chromosomal sequences were incorporated into transgene constructs and these were then introduced into mice. Linking them to the original transgene dramatically enhanced its expression and conferred a degree of position independent expression upon it. However, the results were not as marked when these sequences were linked to other constructs, showing that the effectiveness of such flanking chromosomal sequences is highly dependent on the nature of the transgene used. 相似文献
16.
Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb αS1-casein/human lysozyme DNA construct and a small 5.5 kb β-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born. 相似文献
17.
Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within
a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such
fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements
are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains
stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an “all fish” gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is
regulated by the 5′ promoter and 3′ termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this
study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1α) integrated as a single functional copy into
a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1α integrant was organized
as follows: base pairs 1580–2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete
ocean pout antifreeze 3′ region, and the first 1678 bp of the ocean pout antifreeze 5′ region. Sequence analysis of the EO-1α
integrant and genomic flanking regions in F2 and F4 generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the
consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct.
These results indicate that the EO-1α transgene codes for the chinook salmon GH, and that the transgene and the integration
site have remained stable over multiple generations. 相似文献
18.
Elizabeth A. Maga Gary B. Anderson Mei C. Huang James D. Murray 《Transgenic research》1994,3(1):36-42
Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overal microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5 promoter elements of either the bovine (line B mice) or s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5. 相似文献
19.
Production of transgenic organisms is a well-established, versatile course of action in molecular biology. Genetic engineering often requires heterologous, dominant antibiotic resistance genes that have been used as selectable markers in many species. However, as heterologous 5′ and 3′ flanking sequences often result in very low expression rates, endogenous flanking sequences, especially promoters, are mostly required and are easily obtained in model organisms, but it is much more complicated and time-consuming to get appropriate sequences from less common organisms. In this paper, we show that aminoglycoside 3′-phosphotransferase gene (aphVIII) based constructs with 3′ and 5′ untranslated flanking sequences (including promoters) from the multicellular green alga Volvox work in the unicellular green alga Chlamydomonas and flanking sequences from Chlamydomonas work in Volvox, at least if a low expression rate is compensated by an enforced high gene dosage. This strategy might be useful for all investigators that intend to transform species in which genomic sequences are not available, but sequences from related organisms exist. 相似文献
20.
Tissue-specific, high level expression of the rat whey acidic protein gene in transgenic mice. 总被引:10,自引:0,他引:10
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The importance of intragenic and 3' flanking sequences in the control of the temporal, hormonal and tissue-specific expression of milk whey acidic protein (WAP) has been demonstrated in transgenic mice. Mouse lines carrying a 4.3 kb genomic clone containing the entire rat WAP gene minus 200 bp of the first intron with 0.949 kb of 5' and 1.4 kb of 3' flanking DNA were generated. In eight of nine independent lines of mice analyzed, WAP transgene expression was detected at levels ranging from 1% to 95% (average, 27%) of the endogenous gene. The transgene was expressed preferentially in the mammary gland. Although developmentally regulated during pregnancy and lactation, the temporal pattern of WAP transgene expression differed from the endogenous gene. A precocious increase in expression of the transgene was detected at 7 days of pregnancy, several days earlier in pregnancy than the major increase observed in endogenous mouse WAP mRNA. The rat WAP transgene was translated and secreted into the milk of transgenic mice at levels comparable to the endogenous mouse WAP. This is the first report of a gene that is negatively regulated in dissociated cell cultures as well as in transfected cells, yet is expressed efficiently in the correct multicellular environment of the transgenic mouse. 相似文献