Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100--1800 g ml–1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 g ml–1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein     

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.     

High level expression of the bioactive human interleukin-10 in milk of transgenic mice

Human interleukin-10 (hIL-10) has wide spectrum of anti-inflammatory activities and has shown a potential to be used for treatment of inflammatory or immune illness. In this study, transgenic mice that over-express human interleukin-10 (IL-10) in their milk were generated using a bovine beta-casein/human IL-10 hybrid gene. After cloning of the IL-10 gene, a 22 kb hybrid gene was constructed by linking a 10 kb promoter sequence of the bovine beta-casein gene to the cloned 12 kb IL-10 gene. In six of the eight transgenic mice, the transgene RNA was expressed only in the mammary gland and in the other two mice, it was also slightly expressed in the lung. The highest human IL-10 level in milk was 1620 microg x ml(-1). Notably, transgenes in all the eight transgenic mice were expressed regardless of the integration site even though no correlation was shown between the copy numbers of the transgene and expression level. These results suggest that the genomic sequence of the human IL-10 gene can induce the IL-10 expression at high levels under the control of the bovine beta-casein promoter.     

角质细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有角质细胞特异性角质素5启动子、Cre重组酶基因和人生长激素基因plyA的转基因载体pK5-Cre-hGH。以显微注射的方法将4．2kb的转基因片段K5-Cre-hGH引入小鼠基因组,共注射720枚受精卵,其中龄5枚移植至29只假孕母鼠的输卵管中发育,获得子代小鼠48只,经基因型鉴定有12只小鼠在其基因组上整合有Cre基因,整合率为25％。将带有cre重组酶基因的小鼠与基因组上携带loxP位点的smad4条件基因打靶小鼠杂交以检测Cre重组酶组织特异性表达情况以及介导重组的功能。结果表明,K5-Cre转基因小鼠只在皮肤组织中表达Cre重组酶并能在体内成功地介导loxP位点的重组。     

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo.     

Expression of carbonic anhydrase II (CA II) promoter-reporter fusion genes in multiple tissues of transgenic mice does not replicate normal patterns of expression indicating complexity of CA II regulationin vivo

Although the proximal, 5′ 115 bp of the human carbonic anhydrase II (CA II) gene was sufficient for expression of a reporter gene in some transfected cell lines, we found previously that 1100 bp of this promoter (or 500 bp of the mouse CA II promoter) was not sufficient for expression in transgenic mice. We have now studied the expression of linked reporter genes in mice transgenic for either (1) 11 kb of the human 5′ promoter or (2) 8 kb of the human 5′ promoter with mouse sequences from the first exon, part of the first intron (since a CpG island spans this region), and the 3′ sequences of the gene. Expression was found in both cases, but the tissue specificity was not appropriate for CA II. Although there was a difference in the sensitivity of the assays used, the first construct led to expression in many tissues, while the second construct was expressed only in spleen. These findings indicate considerable complexity of DNA control regions for in vivo CA II expression.     

Human and mouse ABCA1 comparative sequencing and transgenesis studies revealing novel regulatory sequences

The expression of ABCA1, a major participant in apolipoprotein-mediated cholesterol efflux, is regulated by a variety of factors, including intracellular cholesterol concentration. To identify sequences involved in its regulation, we sequenced and compared approximately 200 kb of mouse and human DNA containing the ABCA1 gene. Furthermore, expression of the human gene containing different 5' ends was examined in transgenic mice. Sequence comparison revealed multiple conserved noncoding sequences. The two most highly conserved noncoding elements (CNS1, 88% identity over 498 bp; CNS2, 81% identity over 214 bp) were also highly conserved in other organisms. Mice containing the human ABCA1 gene, 70 kb of upstream DNA, and 35 kb of downstream DNA expressed the transgene similarly to endogenous Abca1. A second transgene beginning 3' to exon 1 was expressed only in liver, providing strong evidence of an unsuspected liver-specific promoter. The identified conserved noncoding sequences invite further investigation to elucidate ABCA1 regulation.     

Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits

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.     

The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice.

Dopamine beta-hydroxylase (DBH) catalyzes the final step in the biosynthesis of norepinephrine, the principal classic neurotransmitter of peripheral sympathetic neurons. We have shown that 5.8 kb of 5' upstream region from a cloned human DBH gene promoter is sufficient to direct expression of the E. coli lacZ gene in transgenic mice to neurons of the locus ceruleus and other classic noradrenergic brain stem nuclei, sympathetic ganglion neurons, and adrenal chromaffin cells. lacZ expression was also observed in neurons of the enteric system, the retina, some sensory and all cranial parasympathetic ganglia, and some diencephalic and telencephalic brain nuclei. The expression pattern of the transgene in DBH-immunonegative sites overlapped with many sites where expression of tyrosine hydroxylase or phenylethanolamine N-methyltransferase, two other catecholamine biosynthetic enzymes, has been reported.     

Analysis of control elements for position‐independent expression of human α‐lactalbumin YAC

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high‐expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human α‐lactalbumin gene express the transgene in a position‐independent manner. The 210 kb YAC was thought to have all the elements necessary for position‐independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the α‐lactalbumin gene had position‐independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position‐dependent expression. Therefore, all the elements necessary for position‐independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the α‐lactalbumin gene. Furthermore, we replaced the human α‐lactalbumin promoter with the bovine αS1‐casein promoter in the 210 kb YAC and produced transgenic rats. Position‐dependent expression was observed. The elements required for position‐independent expression of the bovine αS1‐casein gene are different from those required for the human α‐lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity. Mol. Reprod. Dev. 54:17–23, 1999. © 1999 Wiley‐Liss, Inc.