Rescuing Transgene Expression by Co-Integration

Clark, A. J.; Cowper, Alison E.; Wallace, R.; Wright, Gordon; Simons, Paul

doi:10.1038/nbt1192-1450

Cited by 66 publications

(35 citation statements)

References 18 publications

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“…The production of recombinant fIX in cell culture has proved problematical in the past because many mammalian cell lines are unable to carry out these modifications effectively (12)(13)(14). This laboratory has described transgenic sheep (15) and transgenic mice (10) DNA Constructs. The unmodified bovine ,B-lactoglobulin (BLG) construct, AATD, and FIXD have been described (9,16).…”

mentioning

confidence: 99%

“…In others the segment of DNA encoding the target protein may not be compatible with efficient expression or sequences at the chromosomal site of integration may suppress expression. Attempts have been made to address these problems-for example, by using introns (9) or manipulating the site of transgene integration (10).…”

mentioning

confidence: 99%

“…The unmodified bovine ,B-lactoglobulin (BLG) construct, AATD, and FIXD have been described (9,16). pRT-FIX was constructed by amplifying cDNA from mammary gland RNA from transgenic mouse BIX 33.1, which expresses high levels of a 1450-nt FIXD transcript (10). FIXDA3' was constructed by amplifying a segment of DNA from FIXD containing 5' BLG sequences, the fIX coding sequences, and a short stretch of fIX 3' untranslated region (UTR) lacking the cryptic 3' acceptor site present in FIXD (see Fig.…”

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confidence: 99%

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Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Yull

Harold

Wallace

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.protein in the milk has been very low or undetectable. In this report we show that these low levels are due to the aberrant splicing of the transgene RNA. Removal of the cryptic 3' splice site eliminates this problem, enabling the production of relatively high levels of fIX in the milk of transgenic mice. The purified protein exhibits clotting activity comparable to plasma-derived fIX demonstrating that the mammary gland is capable of the complex post-translational modifications required for biological activity.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Yull

Harold

Wallace

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…The content of hLF in milk of transgenic mice showed some degree of variation between the sequential lactations in a single generation as well as between lactations of different generations (data not shown). This situation is reminiscent of other studies where not all transgenic siblings expressed their transgene to the same level [23]. The possibility that our transgenic line had multiple insertion sites can be excluded because the offsprings had essentially same copy numbers as their founder mice.…”

Section: Discussionmentioning

confidence: 59%

Expression Analysis of a Bovine .BETA.-Casein/Human Lactoferrin Hybrid Gene in Transgenic Mice.

Kim

Lee

et al. 1997

Journal of Reproduction and Development

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Abstract. In our previous study, using a bovine β-casein/human lactoferrin hybrid gene, transgenic mice were generated to target the expression of human lactoferrin (hLF) into the mammary glands. Here, we report the fidelity of transgene expression by eleven transgenic mice and their progeny. Expression of hLF in lactating mice was demonstrated by northern and western blots, ELISA, and immunohistochemical staining. Northern blot analysis in five different tissues revealed that the transgene was expressed in the mammary gland. hLF was expressed in five of eight transgenic mouse lines analyzed by ELISA. The expression level of hLF in milk at day 10 of lactation ranged from 2.5 to 200 µg/ml. The apparent molecular weight of the recombinant hLF was indistinguishable from the native form upon immunoblotting. Immunohistochemical staining revealed that hLF in transgenic mice was exclusively expressed in the epithelial cells of mammary gland. The transgenic mice produced in this study provide a model for the eventual generation of transgenic farm animals producing high levels of hLF in their milk.

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“…Second, the cointegration of constructs containing each of the ", $, and ( encoding sequences for hfib into a transcriptionally responsive chromosomal domain is needed. In some instances, the cointegration of an additional genomic sequence for a milk protein can be used to artificially create a transcriptionally active chromosomal domain for mammary tissue specific expression (Clark et al, 1992;reviewed in Lubon et al, 1996). For the purposes of pharmaceutical production at large-scale, a single cointegration site and associated transgene copy number in that locus is desirable for the facile establishment of a phenotypical and genotypical stable lineage .…”

Section: Transgenic Livestock As An Alternative Source Of Recombinantmentioning

confidence: 99%

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

Butler¹,

O'Sickey²,

Lord³

et al. 1999

Theriogenology

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The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A", B$ and ( fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 :g/ml to 200 :g/ml.In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 :g/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B$ and ( chains were rate limiting. Also, the subunit complexes ( 2 , A"( 2 and the individual subunits A", B$ and ( were found as secretion products. This is the first time that secretion of individual B$-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual B$-chain contained a complex saccharide with low mannose. Glycosylation of the (-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain.iii

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Rescuing Transgene Expression by Co-Integration

Cited by 66 publications

References 18 publications

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Expression Analysis of a Bovine .BETA.-Casein/Human Lactoferrin Hybrid Gene in Transgenic Mice.

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

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