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At least 343 records · Page 19Linked to original sources
PubMed1989

Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA.

Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. We isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

Animals↗
PubMed1997

Cloning of a Chinese hamster ovary (CHO) cDNA encoding phosphatidylserine synthase (PSS) II, overexpression of which suppresses the phosphatidylserine biosynthetic defect of a PSS I-lacking mutant of CHO-K1 cells.

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline. In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant. These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.

Amino Acid Sequence↗
PubMed1997

Expression cloning of rat cDNA encoding UDP-galactose:GD2 beta1,3-galactosyltransferase that determines the expression of GD1b/GM1/GA1.

Using an anti-GD1b monoclonal antibody, expression cloning of a cDNA for the beta1,3-galactosyltransferase gene (EC 2.4.1.62) was performed. KF4C, mouse melanoma B16 transfected with polyoma T antigen gene, and GM2/GD2 synthase cDNA was used as a recipient cell line for the cDNA library transfection. A cDNA clone of GD3 synthase, pD3T-31 was co-transfected with a cDNA library prepared from rat brain RNA using the pcDNAI expression vector. The isolated cDNA clone pM1T-9 predicted a type II membrane protein with 4 amino acids of cytoplasmic domain, 21 amino acids of transmembrane region, and a large catalytic domain with 346 amino acids. Introduction of the cDNA clone into a mouse melanoma line B16 previously transfected with a GM2/GD2 synthase gene resulted in the neo-synthesis of GM1. Co-transfection of the cell line with pM1T-9 and a GD3 synthase cDNA resulted in the expression of GD1b as well as GM1. Moreover, introduction of pM1T-9 into L cell (lacking GM3 synthase), previously transfected with GM2/GD2 synthase gene, resulted in the definite expression of asialo-GM1. These results indicated that GD1b/GM1/GA1 synthases were identical, as previously suggested based on enzymological analysis. In Northern blots of the beta1, 3-galactosyltransferase gene with total RNA from various rat tissues, a 1.6-kilobase mRNA was strongly expressed in spleen, thymus, kidney, and testis. However, the expression level of the gene in the adult brain tissue was not especially high. On the other hand, this gene was expressed at high levels in the rat brain of embryonal day 12, and reached a peak at around birth, then fell to low level in the adult brain.

Aging↗
PubMed1998

Molecular characterization and expression of heparan-sulfate 6-sulfotransferase. Complete cDNA cloning in human and partial cloning in Chinese hamster ovary cells.

Heparan-sulfate 6-sulfotransferase (HS6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-sulfoglucosamine residue of heparan sulfate. The enzyme was purified to apparent homogeneity from the serum-free culture medium of Chinese hamster ovary (CHO) cells (Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol Chem. 270, 4172-4179). From the amino acid sequence data of the purified enzyme, degenerate oligonucleotides were designed and used as primers for the reverse transcriptase-polymerase chain reaction using poly(A)+ RNA from CHO cells as a template. The amplified cDNA fragment was then used as a probe to screen a cDNA library of CHO cells. The cDNA clone thus obtained encoded a partial peptide sequence composed of 236 amino acid residues that included the sequences of six peptides obtained after endoproteinase digestion of the purified enzyme. This cDNA clone was applied to the screening of a human fetal brain cDNA library by cross-hybridization. The isolated cDNA clones contained a whole open reading frame that predicts a type II transmembrane protein composed of 401 amino acid residues. No significant amino acid sequence identity to any other proteins, including heparan-sulfate 2-sulfotransferases, was observed. When the cDNA for the entire coding sequence of the protein was inserted into a eukaryotic expression vector and transfected into COS-7 cells, the HS6ST activity increased 7-fold over the control. The FLAG fusion protein purified by anti-FLAG affinity chromatography showed the HS6ST activity alone. Northern blot analysis revealed the occurrence of a single transcript of 3.9 kilobases in both human fetal brain and CHO cells. The results, together with the ones from our recent cDNA analysis of heparan-sulfate 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985), suggest that at least two different gene products are responsible for 6- and 2-O-sulfations of heparan sulfate.

Amino Acid Sequence↗
PubMed1987

Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein.

We have isolated from a lambda gt11 rat brain cDNA library cDNA clones encoding greater than 95% of the open reading frame and untranslated regions of the mRNA for p38, the most abundant of the integral membrane proteins of the synaptic vesicle. Phage containing cDNA that encoded vesicle proteins were identified by screening fusion proteins with a polyclonal serum to rat brain synaptic vesicles. To identify phage carrying p38 sequences, fusion proteins were used to affinity purify monospecific antibodies from the original heterogeneous serum; antibodies to a 38,000-D protein were then identified by Western blotting. Inserts carrying DNA-encoding p38 sequences were subcloned into plasmid vectors and used to generate cDNA probes for Northern blot analysis. A major transcript of 2.4 kb was expressed specifically in brain and endocrine tissue but not in liver, consistent with the tissue-specific expression of the protein detected by antibody techniques. Using three overlapping clones that encoded fusion proteins, we identified and sequenced approximately 85% of the cDNA. Two additional Eco RI fragments at the 5' end of the mRNA were obtained from a fourth clone identified by screening a second lambda gt11 library with a 5' cDNA probe. The cDNA encoded an open reading frame of 298 amino acids with a 3' untranslated region of 1.4 kb. The protein shares no sequence homology with other Ca2+-binding proteins. The availability of a cDNA clone for an integral synaptic vesicle protein should facilitate studies of its function in transmitter release, its intracellular targeting, and regulation of synaptic vesicle biogenesis during development and regeneration of nerve terminals.

Amino Acid Sequence↗
PubMed1984

Characterization of a cDNA clone for mouse phenobarbital-inducible cytochrome P-450b.

p40, a mouse 1040-nucleotide cDNA clone encoding a form of phenobarbital-inducible cytochrome P-450b, was selected by probing a cDNA library derived from phenobarbital-treated DBA/2N mouse liver with a rat 1830-nucleotide cDNA probe complementary to P-450b mRNA. When rat and mouse liver poly(A)+-enriched total RNAs are probed with p40, control rat P-450b mRNA levels are at least three times as much as control mouse P-450b levels. A 15-fold increase in rat 19S mRNA and only a 3-fold increase in the corresponding mouse 19S mRNA are found following phenobarbital treatment. An intranuclear 4800-nucleotide mRNA precursor is also detectable during the induction process with phenobarbital. Clofibrate, a hypolipidemic drug, is a potent inducer of P-450. There is no cross-hybridization between clofibrate-induced mRNA and either mouse P-450b cDNA or mouse P1-450 cDNA clones. At the level of hybridization stringency used for the Northern analysis, there is no cross-hybridization between phenobarbital-induced mouse or rat P-450b and the P1-450 cDNA probe or between 3-methylcholanthrene-induced mouse or rat P1-450 and the P-450b cDNA probe. These data indicate that the induction process by clofibrate involves activation of P-450 gene(s) not present in the multigene families inducible by either phenobarbital or polycyclic aromatic compounds, i.e., at least three sets of P-450 genes operate independently of one another. Two previously reported cDNA clones of phenobarbital-inducible rat P-450, R17 (P- 450e ) and pcP- 450pb4 (P-450b), were compared with mouse p40.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Optimization of cDNA-AFLP experiments using genomic sequence data.

MOTIVATION: cDNA amplified fragment length polymorphism (cDNA-AFLP) is one of the few genome-wide level expression profiling methods capable of finding genes that have not yet been cloned or even predicted from sequence but have interesting expression patterns under the studied conditions. In cDNA-AFLP, a complex cDNA mixture is divided into small subsets using restriction enzymes and selective PCR. A large cDNA-AFLP experiment can require a substantial amount of resources, such as hundreds of PCR amplifications and gel electrophoresis runs, followed by manual cutting of a large number of bands from the gels. Our aim was to test whether this workload can be reduced by rational design of the experiment. RESULTS: We used the available genomic sequence information to optimize cDNA-AFLP experiments beforehand so that as many transcripts as possible could be profiled with a given amount of resources. Optimization of the selection of both restriction enzymes and selective primers for cDNA-AFLP experiments has not been performed previously. The in silico tests performed suggest that substantial amounts of resources can be saved by the optimization of cDNA-AFLP experiments.

Algorithms↗
PubMed2005

Evaluating and improving cDNA sequence quality with cQC.

SUMMARY: Errors are prevalent in cDNA sequences but the extent to which sequence collections differ in frequencies and types of errors has not been investigated systematically. cDNA quality control, or cQC, was developed to evaluate the quality of cDNA sequence collections and to revise those sequences that differ from a higher quality genomic sequence. After removing rRNA, vector, bacterial insertion sequence and chimeric cDNA contaminants, small-scale nucleotide discrepancies were found in 51% of cDNA sequences from one Arabidopsis cDNA collection, 89% from a second Arabidopsis collection and 75% from a rice collection. These errors created premature termination codons in 4 and 42% of cDNA sequences in the respective Arabidopsis collections and in 7% of the rice cDNA sequences.

5' Untranslated Regions↗
PubMed1980

Sequence complexity of cDNA transcribed from a diverse mRNA population.

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.

Animals↗
PubMed2003

Single primer amplification (SPA) of cDNA for microarray expression analysis.

The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.

Animals↗
PubMed2004

Simple cDNA normalization using kamchatka crab duplex-specific nuclease.

We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

Animals↗
PubMed2005

Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP).

cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5'- and 3'-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT-PCR approaches.

Animals↗
PubMed2001

Signal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines.

Secretions from the esophageal gland cells of plant-parasitic nematodes play critical roles in the nematode-parasitic cycle. A novel method to isolate cDNA encoding putative nematode secretory proteins was developed that utilizes mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages of the soybean cyst nematode Heterodera glycines. The resulting H. glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was transformed into a mutated yeast host for specific selection of cDNA inserts that encode proteins with functional signal peptides. Of the 223 cDNA clones recovered from selection in yeast, 97% of the clones encoded a predicted signal peptide. Fourteen unique cDNA clones hybridized to genomic DNA of H. glycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT II computer analysis. Four cDNA clones hybridized to transcripts within the dorsal esophageal gland cell of parasitic stages of H. glycines, and in situ hybridization within H. glycines was not detected for eight cDNA clones. The protocol provides a direct means to isolate potential plant-parasitic nematode esophageal gland secretory protein genes.

Animals↗
PubMed1988

Molecular cloning of a cDNA for the catalytic subunit of rabbit muscle phosphorylase phosphatase.

A cDNA coding for the catalytic subunit of phosphorylase phosphatase (phosphatase C-I/phosphatase-1c) was cloned from a rabbit muscle cDNA library by screening with oligonucleotide probes. Ten clones were analyzed. The full cDNA sequence of 1395 base pairs contained an open reading frame of 990 base pairs flanked by 3' and 5' noncoding regions of 84 and 321 base pairs, respectively. The DNA sequence (and deduced amino acid sequence) of this cDNA is distinctly different from that of a clone of 1492 base pairs previously reported. Our cDNA is essentially identical to the 1492-base pair clone from residue 182 in the 3' direction, but it is completely different in the 5' direction. Consequently, the amino acid sequence deduced from our cDNA differs by 14 amino acids in the amino terminal from that previously reported and extends for an additional 19 amino acids. Probes to the divergent and common region of our cDNA clone hybridized to an mRNA of the same size by Northern blotting. Thus the cDNA we have isolated appears to code for an isoform of the catalytic subunit of phosphorylase phosphatase.

Amino Acid Sequence↗
PubMed2000

cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora.

For studies of differential gene expression in prokaryotes, methods for synthesizing representative cDNA populations are required. Here, a technique is described for the synthesis of cDNA from the potato pathogens Erwinia carotovora subsp. atroseptica (Eca) and Erwinia carotovora subsp. carotovora (Ecc) using a combination of short oligonucleotide (11-mer) primers that were known to anneal to conserved sequences in the 3' regions of enterobacterial genes. Specific PCR amplifications with primers designed to anneal to 14 known genes from either Eca or Ecc revealed the presence of the corresponding transcripts in cDNA, suggesting that the cDNA represented a broad genomic coverage. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed genes in Eca, including one that shows significant similarity, at the protein level, to an avirulence gene from Xanthomonas campestris pv. raphani. Northern analysis was used to confirm that differentially amplified cDNA fragments were derived from differentially expressed genes. This is the first report of the use of cDNA-AFLP to study differential gene expression in prokaryotes.

Blotting, Northern↗
PubMed2003

beta-alanine N-methyltransferase of Limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant beta-alanine betaine.

Beta-alanine (Ala) betaine, an osmoprotectant suitable under saline and hypoxic environments, is found in most members of the halophytic plant family Plumbaginaceae. In Limonium latifolium (Plumbaginaceae), it is synthesized via methylation of beta-Ala by the action of a trifunctional S-adenosyl L-methionine (Ado-Met): beta-Ala N-methyltransferase (NMTase). Peptide sequences from purified beta-Ala NMTase were used to design primers for reverse transcriptase-PCR, and several cDNA clones were isolated. The 5' end of the cDNA was cloned using a 5'-rapid amplification of cDNA ends protocol. A 500-bp cDNA was used as a probe to screen a lambda-gt10 L. latifolium leaf cDNA library. Partial cDNA clones represented two groups, NMTase A and NMTase B, differing only in their 3'-untranslated regions. The full-length NMTase A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp 5'-untranslated region. The deduced amino acid sequence of 375 residues had motifs known to be involved in the binding of Ado-Met. The NMTase mRNA was expressed in L. latifolium leaves but was absent in Limonium sinuatum, a member of the genus that lacks the synthetic pathway for beta-Ala betaine. NMTase mRNA expression was high in young and mature leaves and was enhanced by light. NMTase cDNA was expressed in yeast (Saccharomyces cerevisiae) under the control of a galactose-inducible promoter. Protein extracts of galactose-induced recombinant yeast had Ado-Met-specific NMTase activities that were highly specific to beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala as methyl acceptors. NMTase activities were not detectable in comparable protein extracts of yeast, transformed with vector control. The NMTase protein sequence shared homology with plant caffeic acid O-methyltransferases and related enzymes. Phylogenetic analyses suggested that beta-Ala NMTase represents a novel family of N-methyltransferases that are evolutionarily related to O-methyltransferases.

Amino Acid Sequence↗
PubMed1996

Retroviral infection with human tyrosinase-related protein-1 (TRP-1) cDNA upregulates tyrosinase activity and melanin synthesis in a TRP-1-deficient melanoma cell line.

A human tyrosinase-related protein-1 (TRP-1) cDNA was inserted into the retroviral vector, pBAbe-puro. Sense and anti-sense constructs were identified and transfected, as well as vector-alone, into a retrovirus packaging cell line by a liposome-mediated technique and used in turn to infect a human melanoma line deficient in TRP-1 protein/transcript. Polymerase chain reaction (PCR) amplification of genomic DNA from these infectants, using TRP-1 cDNA-specific primers, demonstrate that PCR products were only identified from the sense- and anti-sense-infected clones, not from the parental cells or vector-alone infectants. Northern analysis demonstrated that TRP-1 sense and antisense infectants produced TRP-1 cDNA-related transcripts. Immunoblotting analysis with TA99 (a monoclonal antibody for TRP-1) demonstrated a single band of normal molecular weight from melanoma cells infected with sense cDNA, not from cells infected with sense cDNA, not from cells infected with anti-sense or vector-alone, or from the uninfected-parental melanoma cells. The quantitative and qualitative analysis of melanin in the sense and anti-sense infectant cells demonstrated an increase and decrease in pigmentation, respectively, compared with vector alone. Tyrosine hydroxylase and DOPA oxidase activities of tyrosinase hydroxylase and DOPA oxidase activities of tyrosinase were both increased in sense cDNA infected cells plus unaltered or slightly decreased, respectively, in anti-sense cDNA-infected cells compared with control cells. Immunoblotting analysis with anti-tyrosinase antibody (alpha Ty-SP) demonstrated the amount of tyrosinase was slightly increased in TRP-1 overexpressing cells but slightly decreased in anti-sense infectant cells. We have demonstrated that the expression of exogenous TRP-1 cDNA melanoma cells stimulated the activity of tyrosinase and promoted melanogenesis, indicating that TRP-1 plays a role in regulating tyrosinase activity.

Base Sequence↗
PubMedDate not supplied

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.

Animals↗