Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (<Emphasis Type="Italic">Macrozoarces americanus</Emphasis>) antifreeze protein OP5a gene promoter in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5′ region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1α) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1α GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.     

Development of an all-fish gene cassette for gene transfer in aquaculture.

To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.     

Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic Salmon

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.     

High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence.

In our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine beta-casein promoter. The expression level of the protein in milk of 7 mice ranged from 1 to 200 microg/ml; 1 to 34 microg/ml in 6 mice and 200 microg/ml in 1 mouse. With the aim of inducing higher expression of the protein, we constructed an expression cassette comprised of 10 kb of the bovine beta-casein gene promoter and the hLF genomic sequence in place of the cDNA. The hLF genomic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine beta-casein promoter. In total, 8 transgenic mice were generated from 31 mice (transgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hLF expression cassette. Mammary-specific expression of the transgene was addressed by performing Northern hybridization of the total RNAs from various tissues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native protein. The amount of the protein in milk of 5 mice ranged from 60 to 6,600 microg/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500 microg/ml. These data suggest that the genomic lactoferrin sequence represents a valuable element for the efficient expression of the protein in milk of transgenic animals.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

Expression of a Novel Piscine Growth Hormone Gene Results in Growth Enhancement in Transgenic Tilapia (Oreochromis Niloticus)

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallo thionein promoter by exposing fish to increased levels of zinc were also unsuccessful.Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings.The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.     

Cre recombinase-mediated gene targeting of mesenchymal cells

Loss-of-function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the alpha2 chain of collagen type I (Col1alpha2). As an expression vector, we used a P1-derived artificial chromosome (PAC), which harbors approximately 100 kb carrying the col1alpha2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1alpha2. Cre expression was determined by immunohistochemistry and Cre-mediated onset of beta-galactosidase expression in ROSA26R-Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs.     

The extremely high level expression of human serum albumin in the milk of transgenic mice

The recombinant production of human serum albumin has been challenging due to the low unit price and huge amount needed, for the commercial production of rhSA at an economically feasible level, It will be well worth the effort to exploit new method for the extremely high level expression of rhSA. To this end, here a hybrid gene locus strategy was employed, a 37?Kb mWAP-hSA hybrid gene locus was constructed and used as mammary gland specific expression vector, in which the 3?Kb genomic coding sequence in the 24?Kb mouse whey acidic protein (mWAP) gene locus was substituted by the 16?Kb genomic coding sequence of human serum albumin (hSA), exactly from the start codon to the end codon. Corresponding transgenic mice were generated and rhSA was secreted into the milk at an extremely high level of 11.9?g/L. Our transgenic mice carrying the mWAP-hSA hybrid gene locus represent a model system for the cost-effective production of human serum albumin.     

BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice

BOK/MTD was discovered as a protein that binds to the anti-apoptotic Bcl-2 family member MCL-1 and shares extensive amino-acid sequence similarity to BAX and BAK, which are essential for the effector phase of apoptosis. Therefore, and on the basis of its reported expression pattern, BOK is thought to function in a BAX/BAK-like pro-apoptotic manner in female reproductive tissues. In order to determine the function of BOK, we examined its expression in diverse tissues and investigated the consequences of its loss in Bok(-/-) mice. We confirmed that Bok mRNA is prominently expressed in the ovaries and uterus, but also observed that it is present at readily detectable levels in several other tissues such as the brain and myeloid cells. Bok(-/-) mice were produced at the expected Mendelian ratio, appeared outwardly normal and proved fertile. Histological examination revealed that major organs in Bok(-/-) mice displayed no morphological aberrations. Although several human cancers have somatically acquired copy number loss of the Bok gene and BOK is expressed in B lymphoid cells, we found that its deficiency did not accelerate lymphoma development in Eμ-Myc transgenic mice. Collectively, these results indicate that Bok may have a role that largely overlaps with that of other members of the Bcl-2 family, or may have a function restricted to specific stress stimuli and/or tissues.     

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

The biosynthesis of tumor necrosis factor during pregnancy: studies with a CAT reporter transgene and TNF inhibitors.

Tumor necrosis factor (TNF) is a protein hormone which mediates diverse inflammatory conditions, but which also may be involved in physiologic processes. To detect the expression of TNF as it might occur in normal tissues we developed a transgenic mouse line bearing a reporter gene construct in which the INF coding sequence and introns are replaced by a chloramphenicol acetyl transferase (CAT) coding sequence. In these animals, expression of CAT within tissues has been shown to reflect TNF production. We now report upon the pattern of CAT expression that is observed during normal pregnancy. CAT is constitutively expressed in both the fetal and maternal thymuses, and in the placenta, but in no other tissues. Placental CAT activity first becomes measurable at day 13 of gestation, peaks at day 16, and is maintained at high levels until parturition. Crosses between transgenic and non-transgenic mice clearly indicate that the trophoblast, rather than the decidua or uterus, is the source of CAT activity. A soluble TNF receptor/IgG heavy chain chimeric protein, which strongly inhibits TNF activity in vitro and in vivo, was shown to cross the placenta, gaining access to the fetal circulation when administered on the maternal side. However, the chimeric protein did not interrupt pregnancy, and had no obvious effect on fetal development, suggesting that TNF may not be required for completion of a normal gestation.     

Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron

Actin-depolymerizing factor (ADF) is one of the actin cytoskeleton-modulating proteins. We have characterized the accumulation pattern of petunia ADF proteins. PhADF proteins are accumulated in every petunia organ and their accumulation is differentially regulated by developmental signals. Their cellular localization is vascular tissue-preferential in vegetative organs, whereas somewhat different in reproductive organs. In reproductive organs, PhADFs are present in outer integument, endocarp of ovary wall, transmitting tissue of style, and epidermis and endothecium of young anther. From a petunia genomic library, we have isolated a genomic clone encoding PhADF1. Comparison to complementary DNA sequence revealed that the coding region of PhADF1 gene consists of three exons and two introns. Analysis of chimeric gene expression using beta-glucuronidase as a reporter gene in transgenic Arabidopsis revealed that PhADF1 was strongly expressed in every vegetative tissue except petal. In addition, expression of the gene was highly enhanced by its first intron. These results suggest that PhADF1 gene of petunia is mainly expressed in vascular tissues and its expression is regulated by intron-mediated enhancement mechanism.     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.     

E1A基因转导抑制小鼠血管内皮细胞的激活

延缓性排斥反应 (delayedxenograftrejection ,DXR)是进行异种器官移植亟待解决的问题之一 .在DXR过程中 ,核心事件之一是核转录因子NF κB的激活 .人腺病毒 5 (Ad5 )的早期转录产物E1A蛋白可抑制以NF κB为核心的信号转导系统 .利用转基因技术向小鼠的受精卵导入了E1A基因 ,PCR和Southern印迹检测了 4 4只仔鼠 ,其中有 8只整合了E1A基因 .RT PCR检测发现 ,3只小鼠F1代的心、肝、肾等脏器都有E1AmRNA的表达 ,小鼠表型正常 .通过尾静脉向转基因阳性小鼠体内注射人灭活血清 ,模拟DXR发生的生理过程并用免疫荧光检测脏器细胞表面炎症分子E 选择素的表达水平 .结果表明 ,E1A基因的转导显著抑制了小鼠脏器细胞表面E 选择素的表达 ,为解决DXR的发生提供了可行的途径