Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern

Frka, Kosjenka; Facchinello, Nicola; Vecchio, Claudia Del; Carpi, Andrea; Curtarello, Matteo; Venerando, Rina; Angelin, Alessia; Parolin, Cristina; Bernardi, Paolo; Bonaldo, Paolo; Volpin, Dino; Braghetta, Paola; Bressan, Giorgio M.

doi:10.1016/j.jbiotec.2009.02.013

Cited by 10 publications

(11 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We examined the ability of the rtTR‐KRAB Tet‐off repressor construct to regulate gene expression in our system. The Tet‐KRAB system was initially described as a 2‐part system and characterized by transient and stable expression in human cells (29), but the rtTR‐KRAB version of the system has thus far been exclusively used in lentiviral vectors (28, 38). The rtTR‐KRAB repressor construct from pLCVT‐rtTR‐KRAB‐2SM2 was cloned into plasmid expression vectors.…”

Section: Resultsmentioning

confidence: 99%

An adaptable system for improving transposon‐based gene expression in vivo via transient transgene repression

et al. 2013

View full text Add to dashboard Cite

Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.

show abstract

Section: Resultsmentioning

confidence: 99%

An adaptable system for improving transposon‐based gene expression in vivo via transient transgene repression

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Similar good lentiviral transgenesis rates have been achieved in our group also previously [ 16 , 35 ]. When producing transgenic mice via lentiviral transgenesis, each born mouse is unique regarding the transgene copy number and genome insertion point [ 16 , 36 ]. In the produced knockdown mice the transgene copy number was ≈ 3 in F0 generation and declined in F1 generation to ≈ 2 copies on average, which is in line with previous reports on the lentivirally produced transgenic mice [ 35 , 37 ].…”

Section: Discussionmentioning

confidence: 99%

“…The major factor determining the level of target gene silencing is the knockdown transgene copy number [ 36 ]. In addition, in conditional knockdown models, the dosage of gene inducing agent plays a major role.…”

Section: Discussionmentioning

confidence: 99%

“…Dox treatment decreases the infarct size after transient middle cerebral artery occlusion [ 21 ] and reduces long-term cerebral tissue loss and white matter injury after hypoxic-ischemic injury [ 22 ]. Cardioprotective effects of dox after myocardial infarction have also been shown in rats [ 36 ] and in clinical trials, where the dox treatment reduced adverse post-myocardial infarction left ventricular remodelling and also decreased infarct size [ 19 , 20 ].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice

et al. 2018

View full text Add to dashboard Cite

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

show abstract

“…The lentivirus vector was generated as described previously [18], with omission of the concentration step. The vector was classified as a 196b virus and a pLV virus.…”

Section: Methodsmentioning

confidence: 99%

Low Expression of miR-196b Enhances the Expression of BCR-ABL1 and HOXA9 Oncogenes in Chronic Myeloid Leukemogenesis

et al. 2013

View full text Add to dashboard Cite

MicroRNAs (miRNAs) can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05), which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy.

show abstract

Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern

Cited by 10 publications

References 28 publications

An adaptable system for improving transposon‐based gene expression in vivo via transient transgene repression

An adaptable system for improving transposon‐based gene expression in vivo via transient transgene repression

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice

Low Expression of miR-196b Enhances the Expression of BCR-ABL1 and HOXA9 Oncogenes in Chronic Myeloid Leukemogenesis

Contact Info

Product

Resources

About