Low Levels Of Transgene Expression Research Articles

Mutation in Neurofilament Transgene Implicates RNA Processing in the Pathogenesis of Neurodegenerative Disease

A mouse neurofilament light subunit (NF-L) transgene with a 36 bp c-myc insert at the end of the coding region was found to have neuropathic effects on enteric and motor neurons of transgenic mice. The severity of phenotype was related directly to the levels of transgenic mRNA expression. High levels of transgene expression were lethal to newborn pups, causing profound alterations in the development of the enteric nervous system and extensive vacuolar changes in motor neurons. Lower levels of transgene expression led to a transient stunting of growth and focal alterations of enteric and motor neurons. Because the positioning of the c-myc insert coincided with the location of the major stability determinant of the NF-L mRNA (Cañete-Soler et al., 1998a,b), additional studies were undertaken. These studies showed that the c-myc insert alters the ribonucleoprotein (RNP) complexes that bind to the stability determinant and disrupts their ability to regulate the stability of the transcripts. The findings indicate that expression of an NF-L transgene with a mutant mRNA stability determinant is highly disruptive to enteric and motor neurons and implicate alterations in RNA processing in the pathogenesis of a neurodegenerative condition.

Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus.

Tobacco plants were transformed with genes encoding the tobacco vein mottling potyvirus (TVMV) P1 or P3 protein. When compared with vector-transformed or P1 transgenic lines, seedlings of P3 transgenic lines (except a low expressor line) developed poorly, suggesting a detrimental effect of P3 on the plant. All P1 and P3 transgenic lines were protected against the homologous TVMV strain and showed variable proportions of two resistance phenotypes: asymptomatic plants or symptomatic plants that recovered from infection. The resistance was effective against a high inoculum dose but had a narrow spectrum. The heterologous strain TVMV-S was able to overcome resistance in most P1 lines but did not break the resistance of most P3 lines. No line showed resistance to another potyvirus (potato virus Y) or to potato virus X. These features and the low levels of transgene expression in resistant plants suggest that protection in P1 and P3 lines is RNA-mediated. In contrast with most reports on virus-activated gene silencing, some P3 lines with the predominant 'recovery' phenotype showed silencing of the transgene that was activated at a certain developmental stage of the plant independently of virus infection.

Expression of Autoactivated Stromelysin-1 in Mammary Glands of Transgenic Mice Leads to a Reactive Stroma During Early Development

Extracellular matrix and extracellular matrix-degrading matrix metalloproteinases play a key role in interactions between the epithelium and the mesenchyme during mammary gland development and disease. In patients with breast cancer, the mammary mesenchyme undergoes a stromal reaction, the etiology of which is unknown. We previously showed that targeting of an autoactivating mutant of the matrix metalloproteinase stromelysin-1 to mammary epithelia of transgenic mice resulted in reduced mammary function during pregnancy and development of preneoplastic and neoplastic lesions. Here we examine the cascade of alterations before breast tumor formation in the mammary gland stroma once the expression of the stromelysin-1 transgene commences. Beginning in postpubertal virgin animals, low levels of transgene expression in mammary epithelia led to increased expression of endogenous stromelysin-1 in stromal fibroblasts and up-regulation of other matrix metalloproteinases, without basement membrane disruption. These changes were accompanied by the progressive development of a compensatory reactive stroma, characterized by increased collagen content and vascularization in glands from virgin mice. This remodeling of the gland affected epithelial-mesenchymal communication as indicated by inappropriate expression of tenascin-C starting by day 6 of pregnancy. This, together with increased transgene expression, led to basement membrane disruption starting by day 15 of pregnancy. We propose that the highly reactive stroma provides a prelude to breast epithelial tumors observed in these animals.

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

Vector-specific complementation profiles of two independent primary defects in cystic fibrosis airways.

Cystic fibrosis (CF) lung disease has been linked to multiple primary defects in airway epithelia caused by a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) gene. These defects include altered Cl- and Na+ permeability as well as intracellular defects in glycoprotein processing. This apparent diversity in CFTR function is reflected in the complex patterning of CFTR expression in airway epithelia. Such complexities present challenges in the design of CF gene therapies that are capable of reconstituting the endogenous patterns of CFTR gene expression in appropriate target cells. Using a human bronchial xenograft model of the CF airway, we have evaluated the efficacy of recombinant adenoviral and cationic liposome-mediated gene transfer to correct Cl- permeability and mucous sulfation defects found in CF lung disease. Results from these studies demonstrated a clear vector-specific complementation profile for these two defects that was dependent on the type of cell transduced and the level of transgene expression. Single-dose administration of recombinant adenovirus effectively transduced high levels of CFTR transgene expression in 11 +/- 1% of epithelial cells and was capable of correcting cAMP-induced changes in Cl- permeability to 91 +/- 14% that seen in non-CF airways. However, this level of transgene expression was incapable of reversing defects in mucous sulfation due to the lack of efficient targeting to goblet cells. In contrast, cationic liposome-mediated delivery of CFTR encoding plasmids to CF airways achieved extremely low levels of transgene expression with insignificant correction (7.4 +/- 2.4%) of cAMP-induced Cl- permeability. This low level of transgene expression, however, efficiently reduced mucous sulfation to levels seen in non-CF airways. Differences in the complementation profiles of these two vectors in correcting Cl- permeability and mucous sulfation defects mirror the ability of recombinant adenovirus and liposomes to reconstitute only certain features of the endogenous distribution and abundance of CFTR protein expression. Such findings suggest that the level of intracellular CFTR required to facilitate proper glycoprotein processing may be much lower than that needed to mediate bulk Cl- flow across the airway epithelium. In summary, these data present the first example by which two different vector systems can efficiently complement independent primary defects associated with a single dysfunctional gene.

Overexpression of growth hormone affects alternatively spliced IGF-I mRNA expression in oMt1a-oGH transgenic mice.

Restorative growth hormone (GH) treatment of hypophysectomized rats differentially enhances the transcription of alternative IGF-I mRNA classes in liver. The goal of the present study was to determine the effects of GH overexpression on various classes of hepatic IGF-I mRNA in GH transgenic mice. Unstimulated oMt1a-oGH transgenic mice had low levels of transgene expression, and therefore were used to determine the effects of long-term, slightly elevated GH levels on the abundance on each alternative IGF-I mRNA class. The acute effects of high GH levels on the expression of alternative IGF-I mRNA were studied by gavaging transgenic mice with 25 mM zinc sulfate to activate oMt1a-oGH transgene expression. Long-term, low levels of oGH transgene expression in unstimulated transgenic mice resulted in a 73% down regulation of IGF-I 2Ea mRNA but not 1Ea and 2Eb mRNA. Acute stimulation of transgene expression triggered a rapid, 240% increase in 1Ea mRNA levels within 4 hours of transgene expression while 2Ea mRNA was down regulated to nearly non-detectable levels by 6 hours. IGF-I 2Eb mRNA was not affected by the short-term GH elevation. Our results showed that IGF-I 1Ea and 2Ea mRNA were differentially regulated by chronic low or acute high levels of GH. These results suggest that the regulation of IGF-I 1Ea and 2Ea mRNA transcription involve different postreceptor molecules and/or feedback mechanisms.

Enhancement of Adenovirus-Mediated Gene Transfer to Human Bone Marrow Cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1–8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/β-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50–100 resulted in efficient transduction (10.7–15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or β-gal, at a MOI of 50–100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice

BACKGROUND Androgen regulation and prostatic-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (−426 +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing. METHODS To enhance transgene expression, a large fragment of the PB promoter (LPB, −11,500 to +28 bg) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines. RESULTS As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line I, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with glucocorticoids had no signifant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes. CONCLUSIONS This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific and greatly increase levels of transgene expression induced by androgens and zinc. Prostate 32:129–139, 1997. © 1997 Wiley-Liss, Inc.

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice.

Androgen regulation and prostate-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (-426 to +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing. To enhance transgene expression, a large fragment of the PB promoter (LPB, -11,500 to +28 bp) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines. As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line 1, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of CAT increasing from 3 to 40,936 dpm/min/mg protein. Upon sexual maturation and elevated serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with androgens returned LPB-CAT expression to precastration levels. In contrast, treatment with glucocorticoids had no significant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes. This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific expression and greatly increase levels of transgene expression induced by androgens and zinc.

Adenoviral delivery of recombinant DNA into transgenic mice bearing hepatocellular carcinomas.

To evaluate the applicability of recombinant adenoviral vectors in gene transfer to liver cancers, we infused the recombinant adenoviruses AD5CMV-LacZ and Ad5CMV-p53 through the portal veins into two lines of transgenic mice, one bearing the SV40 T antigen and the other the human hepatitis B viral envelope protein. These transgenic animals develop hepatocellular carcinomas (HCC) with predictable pathological manifestations. The levels of expression of the transgenes were dependent upon the viral doses. In all cases, high levels of expression were detected within 2 or 3 days after infusion, but were drastically reduced 7 days after infusion. Significant toxicities were found in the infused animals: > 80% of them died within 7 days after infusion with 10(10) pfu, and transgenic animals bearing HCC apparently were more sensitive to viral toxicity. Although a lower dose (10(9) pfu/animal) produced less toxicity, the levels of expression were substantially reduced (only about 10% of that in animals infused with 10(10) pfu). When Ad5CMV-p53 was infused into animals with nodular hyperplastic stage, the expression of the reporter gene seemed to distribute preferentially at the peripheries of the tumor nodules, and low levels of transgene expression were seen inside the nodules. In tumors in which necrotic lesions were evident, p53 was also expressed at the perpheries of the lesions. These distribution patterns were seen in both tumor models. There was no apparent suppression of tumor growth in the Ad5CMV-p53-infused animals. Our results suggest that alternative methods for gene therapy for HCC need to be explored.

Overexpression of human keratin 16 produces a distinct skin phenotype in transgenic mouse skin.

Human cytokeratin 16 (K16; 48 kDa) is constitutively expressed in postmitotic keratinocytes in a variety of stratified epithelial tissues, but it is best known for the marked enhancement of its expression in stratified squamous epithelia showing hyperproliferation or abnormal differentiation. Of particular interest to us, K16 is strongly induced at the wound edge after injury to the epidermis, and its accumulation correlates spatially and temporally with the onset of reepithelialization. To examine the properties of K16 in its natural cellular context, we introduced a wild-type human K16 gene into the germ line of transgenic mice. Several transgenic lines were established and characterized. Under most conditions, the human K16 transgene is regulated tissue specifically in the skin of transgenic mice. Animals that feature low levels of transgene expression are indistinguishable from controls during the first 6-8 months of life. In contrast, transgenic animals expressing the transgene at higher levels develop skin lesions at 1 week after birth, coinciding with the emergence of fur. At a cellular level, alterations begin with the reorganization of keratin filaments and are first seen at the level of the hair follicle outer root sheath (ORS), where K16 expression is known to occur constitutively. The lesions then progressively spread to involve the proximal epidermis, with which the ORS is contiguous. Elevated transgene expression is associated with a marked thickening of these two epithelia, along with altered keratinocyte cytoarchitecture and aberrant keratinization but no keratinocyte lysis. The implications of this phenotype for epithelial differentiation, human genodermatoses, and wound healing in skin are discussed.

A rat gastrin-human gastrin chimeric transgene directs antral G cell-specific expression in transgenic mice

Gastrin gene expression in the gastrointestinal tract is under both developmental and spatial regulation. In the mature animal, gastrin, an important regulator of parietal acid secretion, is expressed primarily in G cells of the antrum. To determine whether specific promoter elements can direct expression to the gastric antrum in vivo, 450 nucleotides of the proximal rat gastrin promoter were cloned and used to construct a rat gastrin-human gastrin reporter chimeric transgene, which was injected into the mouse germ line. Northern blot analysis, in situ hybridization, and double-label immunocytochemistry studies demonstrated expression of the transgene specifically in antral G cells. Low levels of transgene expression were observed in the ileum and colon, where immunohistochemical studies demonstrated colocalization in enteroendocrine cells expressing peptide YY. The same 450-nucleotide rat gastrin promoter, when joined to the human growth hormone gene, did not result in antral expression. Similarly, a human gastrin-human gastrin reporter transgene also did not achieve antral expression, although it did express in the liver. These results suggest that cis-acting elements present in both the basal 450-nucleotide rat gastrin promoter and the intragenic sequences of the human gastrin gene are necessary to direct expression of a transgene specifically to antral G cells.

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region.

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.

Moloney virus induction of T‐cell lymphomas in a plasmacytomagenic strain of Eμ‐V‐ABL transgenic mice

Although the v-abl gene can provoke several types of lymphoid neoplasm, mice of a transgenic strain (E mu-v-abl 40) in which lymphocytes are targeted for expression of v-abl by a linked immunoglobulin enhancer (E mu) spontaneously develop only plasmacytomas. To determine whether other lymphocytes of this strain were susceptible to transformation, and to identify genes that can collaborate with v-abl in tumorigenesis, E mu-v-abl 40 mice were subjected to insertional mutagenesis by neonatal infection with Moloney murine leukemia virus. Tumorigenesis was accelerated moderately, but nearly all the tumors were T lymphomas. The altered tumor type may reflect both the T-cell tropism of Moloney virus and the higher level of E mu-v-abl 40 expression found in T lymphocytes than in B lymphocytes. Insertion near the c-myc, N-myc or pim-I gene was observed in 42% of the induced tumors, indicating that each of these genes may collaborate with v-abl in lymphomagenesis. Most of the accelerated tumors had a surprisingly low level of transgene expression. Thus, high expression of v-abl may not be required for Moloney-induced T lymphomagenesis.

Wocko, a neurological mutant generated in a transgenic mouse pedigree

Naturally occurring mutations involving the nervous system have provided virtually all of our current understanding of the genetic regulation of neural development (Caviness and Rakic, 1978). The difficulty of isolating the corresponding genes, however, has precluded a molecular analysis of these mutants. Insertional mutagenesis, induced by microinjection of DNA into fertilized ova to produce transgenic animals, provides a molecular tag that marks the site of the mutational event. In this article, we describe a transgenic neurological mutation, designated wocko (Wo), which disrupts the development of the inner ear. These mutant mice display a dominant behavioral phenotype that consists of circling, hyperactivity, and head tossing, reminiscent of the shaker/waltzer class of mutants, and they display a recessive homozygous sublethal phenotype. Anatomical analyses showed that both structural and neural components of the vestibular system were disrupted, while analyses of mutant fetuses showed that these morphological abnormalities were due to aberrant development. Although low levels of transgene expression were detected using a sensitive PCR assay, several nonmutant pedigrees that contain the same construct also expressed the transgene in the inner ear, suggesting that low levels of transgene expression alone were not responsible for the wocko phenotype. Because the integrated transgene provides a marker to clone the wocko mutation, the analysis of this mutant will give unique insight into the molecular genetics of inner ear development and into a broad class of neurological mutations that affect the inner ear.