Insulin-like Growth factor-I cDNA Research Articles

Molecular cloning, expression analysis of insulin-like growth factor I (IGF-I) gene and IGF-I serum concentration in female and male Tongue sole (Cynoglossus semilaevis)

Insulin-like growth factor I (IGF-I) is a polypeptide hormone that regulates growth during all stages of development in vertebrates. To examine the mechanisms of the sexual growth dimorphism in the Tongue sole (Cynoglossus semilaevis), molecular cloning, expression analysis of IGF-I gene and IGF-I serum concentration analysis were performed. As a result, the IGF-I cDNA sequence is 911 bp, which contains an open reading frame (ORF) of 564 bp encoding a protein of 187 amino acids. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. The IGF-I mRNA was predominantly expressed in liver, and the IGF-I expression levels in females and extra-large males were 1.9 and 10.2 times as much as those in normal males, respectively. Sex differences in IGF-I mRNA expressions at early life stages were also examined by using a full-sib family of C. semilaevis, and the IGF-I mRNA was detected at all of the 27 sampling points from 10 to 410 days old. An increase in IGF-I mRNA was detected after 190 day old fish. The significantly higher levels of IGF-I mRNA in females were observed after 190 days old in comparison with males (P<0.01). The IGF-I concentrations in serum of mature individuals were detected by ELISA. The IGF-I level in the serum of females was approximately two times as much as that of males. Consequently, IGF-I may play an important role in the endocrine regulation of the sexually dimorphic growth of C. semilaevis.

Insulin‐like growth factor‐I of pejerrey, Odontesthes bonariensis: cDNA characterization, tissue distribution and expression profiles after growth hormone administration

The liver production of the insulin-like growth factor-I (IGF-I) is a key factor in the endocrine control of body growth by a growth hormone. As pejerrey Odontesthes bonariensis has been reported as a fish with low growth rates in captivity, basic research on this respect is needed in order to understand it. In this context, the pejerrey IGF-I cDNA was cloned and its hepatic expression was examined in fish after recombinant pejerrey growth hormone (pjGHr) administration. The full length of IGF-I transcript showed a high sequence similarity to other teleost sequences. The tissue distribution analysis by reverse transcriptase polymerase chain reaction in adult fish revealed that IGF-I expressed ubiquitously with the highest mRNA levels in the liver, posterior intestine and brain. No alternative IGF-I mRNA was found in the liver, as it was reported for other teleosts. IGF-I transcript was measured in the liver after pjGHr in vivo stimulation by means of quantitative real-time polymerase chain reaction assays. A dose-dependent response of IGF-I mRNA was observed after pjGHr administration, and reached a six-fold IGF-I maximum increase over control group when 2.5 microg pjGH/g-body weight (bw) was injected. Temporal analysis of hepatic IGF-I mRNA level showed that administration of a single dose of pjGHr into juvenile pejerrey resulted in a significant increase (P <0.02) 9 hours post-injection (hpi). These results add novel information on the nucleotide sequence of IGF-I in Atheriniformes and demonstrate that pjGHr could promote a dramatic response in liver, increasing the IGF-I mRNA level.

The combination of IGF-I and KGF cDNA improves dermal and epidermal regeneration by increased VEGF expression and neovascularization

Insulin-like growth factor-I (IGF-I) and keratinocyte growth factor (KGF) cDNA gene transfer individually improves dermal and epidermal regeneration. The aim of the present study was to determine whether the combination of IGF-I plus KGF cDNA further improves wound healing and by which mechanisms these changes occur. Rats received an acute wound and were divided into four groups to receive weekly subcutaneous injections of liposomes plus Lac Z cDNA, liposomes plus IGF-I cDNA, liposomes plus KGF cDNA, or liposomes plus IGF-I/KGF cDNA. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine IGF-I, KGF, platelet-derived growth factor, fibroblast growth factor (FGF), transforming growth factor-beta and vascular endothelial growth factor (VEGF) expression and different types of collagen (I, III and IV). IGF-I, KGF and their combination cDNA treatment significantly (P<0.05) accelerated re-epithelization, increased IGF-I, KGF, FGF, VEGF and collagen type IV expression, while it had no effect on collagen type I and III expression. The combination of IGF-I plus KGF cDNA increased (P<0.05) neovascularization and VEGF expression when compared to IGF-I cDNA, KGF cDNA groups and controls. In conclusion, exogenous administration of liposomal IGF-I plus KGF cDNA enhanced dermal and epidermal regeneration which is due to increased neovascularization.

Cloning, Characterization and Tissue Specific Expression of Amur Tiger (Panthera tigris altaica) IGF-I

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

Expression of recombinant triangular bream (Megalobrama terminalis) pre-IGF-I in Escherichia coli

The insulin-like growth factor I (IGF-I) cDNA (GenBank Accession No. AY247412) of triangular bream (Megalobrama terminalis) was expressed for the first time in Escherichia coli. To construct the expression plasmid, the IGF-I cDNA was subcloned into prokaryotic-expressing vector pGEX-4T-1. The E.coli JM109 was transformed with the recombinant plasmid pGEX-4T-1-IGF-I, and the transgene expression was observed after being induced with isopropyl-β-D-thiogalactoside (IPTG). The results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting indicated that the recombinant fusion protein had immune activity and the molecular weight was about 47 kDa. The results of SDS-PAGE and thin layer scanning showed that the yield of fusion protein had been enlarged with prolonging time. When the time of induced expression was 1, 2, 3, 4, 5, and 6 h, the expression amount was approximately 1.4, 4.3, 8.1, 11.3, 16.3 and 18.8% of total bacterial protein respectively.

Insulin-like Growth Factor-1 Gene Transfer Augments Muscle IGF Protein Content Despite Cast Immobilization

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that has been shown to play a critical role in muscle regeneration and muscle hypertrophy. Viral-mediated gene transfer of IGF-I appears to facilitate muscle hypertrophy and improve muscle function, yet it is unclear whether the benefits of gene transfer are maintained during cast immobilization or with subsequent restoration of muscle function following cast immobilization. PURPOSE: To investigate the potential of virus-mediated gene transfer of IGF-1 to augment IGF-1 protein levels in skeletal muscle under normal loading/activity conditions and whether elevations are maintained during and after cast immobilization with subsequent reloading. METHODS: The anterior compartments of the left hindlimb of 43 young adult C57BL6 mice were injected with a recombinant adeno-associated virus vector for IGF-I (rAAV-1). The construct consisted of the entire rat IGF-I cDNA which encodes for IGF-I, a Myosin Light Chain (MLC) 1/3 promoter and enhancer, and SV40 polyadenlyation sequence. At 20 weeks of age, 35 of these mice were immobilized in a bilateral cast for 2 weeks with ∼60° of dorsiflexion to maximize anterior compartment muscle atrophy. After cast immobilization, the tibialis anterior muscle was removed from both legs at 0 days and 1,3,5, and 10 weeks of free cage reambulation. Eight mice remained uncasted for comparison. RT-PCr was performed to quantify IGF-1 mRNA levels. IGF-1 protein levels were quantified using a mouse IGF-1 immunoassay. RESULTS: IGF-1 mRNA levels were consistently elevated in injected limbs. In addition, there was a 27 fold increase in tibialis anterior IGF-1 protein content of the injected limb in uncasted mice (85.2+32.9ng/g injected; 3.2+0.48ng/g uninjected) (p<0.05). Significant differences in absolute values of IGF-1 protein content from injected to uninjected limbs persisted with cast immobilization and free cage reambulation (range: 14–19 fold differences) (p<0.05). When comparing the relative magnitude of side-to-side differences in IGF protein content across all timepoints, there was a significantly greater difference in IGF-1 protein content before cast immobilization (p<0.05). CONCLUSION: Muscle specific adeno-associated virus overexpression of IGF-I not only increased IGF-1 mRNA levels, but also resulted in markedly higher IGF-1 protein levels that remained elevated despite cast immobilization. Our findings suggest that gene transfer of IGF-1 may be able to guard skeletal muscle from the deleterious impact of immobilization/disuse and enhance the subsequent restoration of muscle function.

Interaction of exogenous liposomal insulin‐like growth factor‐I cDNA gene transfer with growth factors on collagen expression in acute wounds

Growth factors have been shown to modulate the complex cascade of wound healing, however, interaction between different growth factors during dermal and epidermal regeneration is still not entirely defined. We have recently shown that exogenous liposomal gene transfer of cDNA results in physiologic expression and response in an acute wound. In the present study we determined the interaction between insulin-like growth factor-I (IGF-I), a mesenchymal growth factor, administered as liposomal cDNA, with other dermal and epidermal growth factors on collagen synthesis in an acute wound. Sprague-Dawley rats were given a scald burn to inflict an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus a beta-galactosidase containing plasmid (Lac Z [0.2 microg, vehicle]), or liposomes plus the IGF-I cDNA containing plasmid (2.2 microg) and Lac Z (0.2 microg). Immunological assays, histological and immunohistochemical techniques were used to determine growth factor concentration and different types of collagen (I, III, and IV) after IGF-I cDNA gene transfer. IGF-I cDNA transfer accelerated reepithelization and was associated with increased levels of IGF-I, fibroblast growth factor, keratinocyte growth factor, vascular endothelial cell growth factor, and platelet-derived growth factor protein expression. IGF-I cDNA had no effect on transforming growth factor-beta. IGF-I cDNA significantly increased type IV collagen while it had no effect on types I and III collagen. Exogenously administered IGF-I cDNA increased protein concentrations of keratinocyte growth factor, fibroblast growth factor, platelet-derived growth factor, and type IV collagen. We conclude that liposomal IGF-I gene transfer can accelerate wound healing without causing an increase in types I and III collagen expression.

Insulin-like Growth Factor-1 Gene Transfer Augments Muscle Protein Synthesis Rate And Size In Adult Mice

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that has been shown to play a critical role in muscle regeneration and muscle hypertrophy. Viral-mediated gene transfer of IGF-I upregulates muscle size and increases muscle strength. It is not clear whether IGF-I-induced muscle hypertrophy is mediated by an increase in protein synthesis and/or a decrease in proteolysis rates in existing myofibers and satellite cells. PURPOSE The objective of this study was to investigate the effect of IGF-I overexpression on in vivo muscle protein synthesis rate. METHODS The left hindlimb of young adult C57BL6 mice was injected with a recombinant adeno-associated virus vector for IGF-I (rAAV-1). The construct consisted of the entire rat IGF-I cDNA which encodes for IGF-I, a Myosin Light Chain (MLC) 1/3 promoter and enhancer, and SV40 polyadenlyation sequence. Six mice (age=3 weeks) were injected with 80ul of 10% glycerol/PBS containing approximately 1010 rAAV particles into the interstitial space of the anterior compartment of one hindlimb, targeting the extensor digitorum longus (EDL). Three months after transfection, in vivo mixed muscle protein synthesis rates were measured using the incorporation of intravenously administered L- [13C]-phe into EDL muscle proteins. [13C]-phe abundance in the structural proteins was quantitated using gas chromatography-combustion-isotope ratio mass spectrometry. Paired samples t-tests were used for comparisons between transfected and contralateral hindlimbs. RESULTS rAAV-IGF1 transfection resulted in a significant increase (mean ± SD) (12.1 ± 8.5%) in muscle wet weight and cross-sectional area (9.9 ± 9.1%)(p<0.05). In vivo mixed muscle protein synthesis rate was 26.9 ± 15.6% higher in the rAAV-IGF1 injected limbs compared to the contralateral control limb (p<0.05). Average EDL muscle protein synthesis rate was 10.5 ± 2.8%/day in the rAAV-IGF1 injected limb and 8.2 ± 1.9%/day in the contralateral limb. CONCLUSIONS These results demonstrate that muscle specific adeno-associated virus overexpression of IGF-I increased mixed muscle protein synthesis rate and this contributed to muscle hypertrophy. Further research will determine EDL contractile properties, and the components of the IGF-I signaling pathway that are activated in satellite cells and mature myofibers. Supported by R01-HD042955, NCRR-000954.

Effects of estriol on proliferative activity and expression of insulin-like growth factor-I (IGF-I) and IGF-I receptor mRNA in cultured human osteoblast-like osteosarcoma cells

The present study was undertaken to elucidate whether estriol (E3) affects the proliferative activity and the expression of insulin-like growth factor-I (IGF-I) mRNA and IGF-I receptor (IGF-IR) mRNA in cultured human osteoblast-like osteosarcoma cells (HOS TE85). In this study, the effects of E3 on cultured HOS TE85 cells were compared with those of 17β-estradiol (E2). HOS TE85 cells were subcultured in phenol red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 72 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of E3 (3.52 × 10−9, 3.52 × 10−8, 3.52 × 10−7 mol/l) or E2 (3.67 × 10−8 mol/l). Treatment with either E3 (3.52 × 10−8, 3.52 × 10−7 mol/l) or E2 (3.67 × 10−8 mol/l) resulted in an increase in the number of cultured HOS TE85 cells and their uptake of bromodeoxyuridine. Northern blot hybridization with a IGF-I cDNA probe revealed that RNA prepared from cultured HOS TE85 cells contained IGF-I mRNA transcripts of 1.8, 4.4 and 7.5 kb. Treatment with either E3 (3.52 × 10−9, 3.52 × 10−8, 3.52 × 10−7 mol/l) or E2 (3.67 × 10−8 mol/l) resulted in increased expression of the three mRNA transcripts relative tot hose in untreated control cultures. Semi-quantitative, reverse transcription polymerase chain reaction analysis showed that the 440-bp IGF-IR mRNA transcript was present in HOS TE85 cells and that treatment with either E3 or E2 did not affect the IGF-IR mRNA expression in these cells. These results demonstrate that E3 (3.52 × 10−9, 3.52 × 10−8, 3.52 × 10−7 mol/l) exerts profound effects on the proliferative potential of cultured HOS TE85 cells, compatible with that of E2 (3.67 × 10−8 mol/l), through the induction of IGF-I mRNA expression without affecting IGF-IR mRNA expression in these cells.

Cellular and physiologic effects of exogenous liposomal IGF-1 cDNA gene transfer in acute wounds

The purpose of the present study was to examine the molecular interactions after liposomal cDNA gene transfer and whether the exogenous gene transfer is recognized by the cell and causes endogenous cellular and physiologic responses. Insulin-like growth factor-I (IGF-I) is known to cause, when administered as a protein, adverse side effects, due to lack of cellular responses. Therefore, we used IGF-I cDNA as a vector to study cellular and physiological effects after liposomal administration in an acute wound. Sprague-Dawley rats were inflicted an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.2 μg, vehicle), or liposomes plus the IGF-I cDNA (2.2 μg) and Lac Z gene (0.22 μg). Transfection was confirmed by histochemical assays for β-galactosidase. Planimetry, immunological assays, and histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, and dermal and epidermal regeneration. IGF-I cDNA transfer increased IGF-I protein expression and caused concomitant cellular responses by increasing IGFBP-3 and decreasing IGFBP-1. IGF-I cDNA transfer increased IGF-I, FGF, KGF, and PDGF protein concentration, while it had no effect on TGF-β. IGF-I cDNA exerted promitogenic anti-apoptotic effects on basal keratinocytes, thus improving epidermal regeneration. IGF-I cDNA improved dermal regeneration by an increased collagen IV deposition. IGF-I cDNA increased VEGF concentrations and thus neovascularization. Exogenous administered IGF-I cDNA is recognized by the cell and leads to similar intracellular responses as the endogenous gene. Liposomal IGF-I gene transfer further leads to improved dermal and epidermal regeneration by interacting with other growth factors.

Hepatic mRNA expression and plasma levels of insulin-like growth factor-I (IGF-I) in broiler chickens selected for different growth rates

The hepatic expression and plasma concentrations of IGF-I were investigated in three broiler chicken strains selected for different growth rates (HP-Hubbard-Pettersen, a fast growing strain; NN-Naked-neck, a strain with an intermediate growth rate and a heterozygous genotype, and C-Caipira, a slow growing crossbred strain). The chickens were studied at 1, 21 and 42 days of age and had free access to food throughout the study. Hepatic IGF-I mRNA expression was assessed by dot blot analysis using a randomly labeled chicken IGF-I cDNA as the probe and plasma IGF-I concentrations were assayed by radioimmunoassay. The hepatic levels of IGF-I mRNA increased from 1 to 21 days of age in all strains, with NN chickens showing a higher (p < 0.05) IGF-I expression than the other strains. Plasma IGF-I concentrations increased (p < 0.05) with broiler chicken age, but there were no significant differences among the strains. These results indicate that despite differences in the growth rates among the strains, the changes in the expression of IGF-I mRNA in liver and in the plasma levels of IGF-I were independent of broiler chicken strain, but varied with chicken age.

Common carp insulin-like growth factor-I gene: complete nucleotide sequence and functional characterization of the 5′-flanking region

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of fish. To understand the molecular mechanism which controls the transcription of the IGF-I gene in common carp, we have cloned and completely sequenced the IGF-I gene and the 5′-flanking region from a local tropical fish, the common carp ( Cyprinus carpio), and characterized its promoter activity by transfection into human embryonic kidney (293GHR) cells which express the human growth hormone (GH) receptor. The common carp gene is the smallest IGF-I gene known so far, spanning approximately 13 kb, and is consisted of five exons and four introns. The sequence of the gene is consistent with the single type of IGF-I cDNA that we have isolated previously from a common carp liver cDNA library. The expression pattern of IGF-I is similar between juvenile carp and adult carp. While liver was found to be the major site of IGF-I gene expression in common carp, the expression levels in other tissues are relatively low. Like many other IGF-I gene promoters, there are no apparent TATA box and CCAAT box upstream of the transcription initiation site. However, sequence analysis of the common carp IGF-I promoter region identified several consensus liver-enriched transcription factor binding sites, including HNF-1α, HNF-3β, C/EBP, and one STAT5. We have analyzed the promoter activity of the 5′-flanking region of the common carp IGF-I gene by performing luciferase reporter assays in transfected 293GHR cells. Addition of human GH to the transfected cells led to an increased expression of the reporter gene, indicating that the cloned genomic fragment possessed promoter activity. This was confirmed by the lack of promoter activity of a construct in which the putative promoter was cloned in a reverse orientation upstream of the reporter gene. The liver-specific transcription factor, hepatic nuclear factor (HNF)-1α, was also found to be involved in the regulation of the common carp IGF-I transcription. Transfection results from a set of deletion mutants helped to map the locations of the responsive elements on the promoter responsible for the GH effect and for the interaction with HNF-1α. These observations provide information for delineating the transcriptional regulation of IGF-I gene expression in common carp.

Cloning and characterization of the bovine class 1 and class 2 insulin-like growth factor-I mRNAs

Insulin-like growth factor-I (IGF-I) is an important regulator of growth, development, and metabolism, and is the primary mediator of the growth-promoting activity of growth hormone (GH) in animals. In several species, the IGF-I polypeptide is generated from IGF-I mRNA containing either exon 1 (class 1 IGF-I mRNA) or exon 2 (class 2 IGF-I mRNA) as the leader exon. The objectives of this study were to identify class 1 and class 2 IGF-I mRNAs in cattle and to compare their expression in different tissues, their response to GH, and their translational efficiency. Three class 1 IGF-I cDNAs corresponding to three different transcription start sites in exon 1 and one class 2 IGF-I cDNA were identified from adult cattle liver using 5′-rapid amplification of cDNA ends (5′-RACE). Both classes of IGF-I mRNAs were expressed in a variety of tissues, with the highest level in liver; class 1 IGF-I mRNA was more abundant than class 2 IGF-I mRNA in all tissues. Six hours after a single intramuscular injection of 500 mg of recombinant bovine GH, class 1 and class 2 IGF-I mRNAs in steer liver were increased by 29% ( P=0.07) and 62% ( P<0.05), respectively. The luciferase reporter mRNA fused to a class 1 IGF-I 5′-untranslated region (5′-UTR) was translated four times more efficiently in vitro than the luciferase reporter mRNA fused to a class 2 IGF-I 5′-UTR ( P<0.05). These results indicate that the IGF-I gene in cattle is transcribed as class 1 and class 2 IGF-I mRNAs and that the two classes of IGF-I mRNAs may be regulated differentially at both transcriptional and translational levels.

Ovarian expression of human insulin-like growth factor-I in transgenic mice results in cyst formation.

Insulin-like growth factor-I (IGF-I) has been implicated in a wide variety of physiological processes including ovarian function. To better understand the ovarian role of IGF-I, transgenic mice harbouring a human IGF-I cDNA (hIGF-I) under the control of the mouse LH receptor promoter were generated. Expression of the hIGF-I, determined by Northern blot, was found to occur in the gonad tissues of these transgenic mice. The hIGF-I protein was also detectable by radioimmunoassay in ovarian extracts as well as in the plasma. The fertility of mating transgenic females, as estimated by the number of implantation sites post-coitum, did not appear to be affected. However, transgenic females who failed to mate and produce offspring were found to possess polycystic ovaries. Evaluation of testosterone, estradiol, and LH levels revealed that transgenic animals had significantly elevated circulating levels of testosterone compared to their non-transgenic littermates, while LH levels in transgenic females were significantly lower. Yet, estradiol appeared to be unaffected. These results support the contention that the IGF system plays an important role in ovarian function and that an imbalance in this system may result in ovarian pathology.

Biodistribution and Feasibility of Non-Viral IGF-I Gene Transfers in Thermally Injured Skin

Gene therapy using cationic liposomes containing cDNA is a relatively new approach with great potential; however, little is known about the mechanisms of dermal gene transfer, its biodistribution, systemic transfection, and cellular uptake. This study identifies mechanisms, transfection rates, and biodistribution of liposomal gene transfers in the skin of thermally injured rats using cDNA gene constructs coding for insulin-like growth factor-I (IGF-I) and Lac Z. Male Sprague-Dawley rats (350 to 375 g) were given a 60% total body surface area full-thickness scald burn that was followed by weekly subcutaneous injections of normal saline (control, n = 10), liposomes plus 0.2 μg Lac Z cDNA construct driven by a cytomegalovirus (CMV) promoter (vehicle, n = 10), or liposomes containing 2.2 μg cDNA coding for IGF-I plus 0.2 μg Lac Z cDNA construct driven by a CMV promoter (IGF-I cDNA, n = 10). Gene transfection was determined by histochemical and luminescent β-galactosidase assays of blood, skin, liver, spleen, and kidney. Transcription of IGF-I cDNA to IGF-I mRNA was determined in skin cells by Northern blot analyses. Levels of IGF-I protein in blood, skin, liver, spleen, and kidney were measured by radioimmunoassay. The biological activity of the translated IGF-I was evaluated by the mitogenic activity in dermal cells and the rate of re-epithelization. Gene transfection was observed only in skin cells. The expression of IGF-I mRNA increased in skin cells of burned rats receiving liposomes containing the IGF-I cDNA construct compared with liposomes without the construct or normal saline. IGF-I protein levels in the skin of rats receiving the IGF-I cDNA was 176 ± 4 ng/ml compared with 105 ± 6 ng/ml for liposomes alone or 90 ± 3 ng/ml for saline (p < 0.05). The translated IGF-I protein was found biologically active in the skin by increasing skin cell proliferation and accelerating re-epithelization 33 days after thermal injury (p < 0.05). No systemic transfection could be detected. Skin cells transfected with liposomes encapsulating the IGF-I cDNA constructs increased the expression of IGF-I mRNA transcript and the expression of a biologically active IGF-I protein. Liposomes containing the cDNA coding for IGF-I present an effective approach to gene therapy in the skin.

Insulin-like growth factor-I cDNA gene transfer in vitro and in vivo.

Our hypothesis is that gene transfer of an IGF-I CMV-cDNA with cholesterol containing cationic liposomes is an efficient tool for transient transfection of growth factors in vitro and in vivo. In vitro, we transiently cotransfected IGF-I cDNA with a CMV construct and a Lac Z beta-galactosidase cDNA/CMV construct using cholesterol containing cationic liposomes and measured beta-galactosidase and IGF-I mRNA and protein. In vivo, we subcutaneously injected 3-month-old male Sprague-Dawley rats with IGF-I cDNA and beta-galactosidase cDNA into rat skin. After IGF-I and beta-galactosidase were cotransfected into PC12 cells, Northern blot analysis showed that the peak time of IGF-I expression was 2 days for mRNA and 5 days for protein. In vivo, a cDNA/liposome ratio of 1:2 was most effective. IGF-I protein expression in IGF-I-transfected skin resulted in significant transfection from day 5 to day 7. In situ determination of beta-galactosidase activity confirmed that transfections resulted in a restricted expression area.

Effect of multiple gene transfers of insulinlike growth factor I complementary DNA gene constructs in rats after thermal injury.

Multiple subcutaneous injections of cholesterol-containing cationic liposomes encapsulating the complementary DNA (cDNA) gene for insulinlike growth factor I (IGF-I) increase the rate of transfected skin cells and result in increased IGF-I protein levels in the skin with subsequent improvement in wound healing when compared with a single injection. Laboratory. Twenty-four adult male Sprague-Dawley rats (350-375 g) received a full-thickness scald burn on 60% of their body surface. These rats were randomly divided to receive either 1 injection of liposomes containing 2.2 microg-cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct, or 2 injections of liposomes containing 2.2 microg cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct. Transfection rates and IGF-I protein levels in the skin and physiological responses to the IGF-I gene therapy, evaluated from changes in body weight, protein content in serum and liver, and the rate of burn wound healing. There was a significant decrease in transfection rate and IGF-I protein expression distal from the injection site in animals receiving 1 injection, as compared with a consistent increase in rats receiving multiple injections. Multiple injections improved the response to thermal trauma by increasing the extent of the healed burn wound 33 days after thermal injury (single injection, 31% +/- 1% vs multiple injections, 38% +/- 2%), total serum protein (single injection, 52 +/- 0.5 g/L vs multiple injections, 55 +/- 0.6 g/L), and total liver protein (single injection, 82.0 +/- 0.3 mg/mL vs multiple injections, 91.0 +/- 3.8 mg/mL), P<.05. Gene transfer rates can be increased by multiple injections of liposomes encapsulating IGF-I cDNA constructs. Increased transfer results in greater IGF-I protein skin concentrations, accelerated wound healing, and increased serum and liver protein concentrations. The clinical relevance of these findings is that liposomal gene constructs should be applied in well-defined distances to improve gene transfer in the skin, and thus clinical outcome after thermal injury.

Testes-specific transgene expression in insulin-like growth factor-I transgenic mice.

Insulin-like growth factor-I (IGF-I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF-I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF-I over-expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF-I cDNA (hIGF-I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF-I into a variety of tissues both reproductive and non-reproductive. Yet expression of the foreign hIGF-I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV-hIGF-I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF-I serum levels, growth rates, or testicular histology have been observed between transgenic and non-transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV-hIGF-I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP-2 and IGFBP-3. These results suggest that the testicular over-expression of IGF-I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV-hIGF-I transgenic mice, despite the presence of elevated testicular IGF-I. The observation that testis specific IGF-I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function.

IGF-I gene transfer in thermally injured rats.

Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for beta-galactosidase, liposomes plus cDNA for IGF-I and beta-galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for beta-galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). Local IGF-I protein concentrations in the skin were higher when compared to liposomes containing only the lacZ gene (P < 0.05) Transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. Liposomes containing the IGF-I gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.

Induction of apoptosis in rat hepatocarcinoma cells by expression of IGF-I antisense c-DNA

Background/Aims: We have developed a gene therapy strategy based on the observation that insulin-like growth factor I (IGF-I) is necessary for the acquisition and maintenance of the transformed phenotype in hepatocarcinoma. This strategy consists in transfecting the rat hepatoma cell line with an episomal vector expressing the antisense IGF-I cDNA under the control of the metallothionein I promoter inducible by zinc, decreasing therefore the level of IGF-I in these cells. The transfected clones lost their tumorigenic properties, and were able to induce, in vivo, the regression of an established tumor in syngeneic rats. To understand the loss of tumorigenic properties of these transfected clones, we have quantified, by different approaches the number of apoptotic cells according to the level of IGF-I expression. Methods: IGF-I antisense synthesis is transfected cells was stimulated using zinc. We then characterized and quantified apoptosis, in these transfected clones, by morphological and DNA fragmentation analyses, flow cytometry and comet assay. Results: We have demonstrated that IGF-I inhibits the development of apoptosis in parental cells, that the transfected clones are able to restore the spontaneous apoptotic programme, and that apoptosis increases massively when overexpression of IGF-I antisense is caused by zinc stimulation of the metallothionein I promoter. Conclusion: The present results allow us to conclude that the level of apoptotic pathway in liver cell lines is directly related to the amount of IGF-I deficiency.