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A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

Espana-Agusti J, Tuveson DA, Adams DJ, Matakidou A - Sci Rep (2015)

Bottom Line: The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation.We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection.Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Cambridge, CRUK Cambridge institute, Cambridge, CB2 0RE, UK.

ABSTRACT
The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.

No MeSH data available.


Related in: MedlinePlus

Efficient and sustained renal tubular gene delivery via Ultrasound-guided intraparenchymal injection of lentiviral vectors.(a) Diagram of pELS lentiviral construct. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine tract; EF1, elongation factor 1 alpha promoter; E2A, self-cleaving 2A peptide; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. (b) Representative ultrasound images of left renal parenchymal injections in adult mice. White arrow shows kidney location, arrow tip points to the injection needle. (c) Whole body bioluminescence of kidney-specific luciferase expression in representative C57/BL6J mice that underwent ultrasound-guided left intrarenal injection of either ELS or control (no luciferase) lentiviral vector (left hand mouse) at 7 days post injection. The relative luminescence intensity is indicated by a colour scale bar. (d) Representative immunofluorescent images of renal sections of adult (top panels) and neonatal (bottom panels) ELS injected and control (uninjected) kidneys, 60 days post infection. Strawberry expression was limited to the renal cortex and corticomedullary junction. Cor, cortex; Med, medulla. Scale bars, 50 μm.
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f1: Efficient and sustained renal tubular gene delivery via Ultrasound-guided intraparenchymal injection of lentiviral vectors.(a) Diagram of pELS lentiviral construct. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine tract; EF1, elongation factor 1 alpha promoter; E2A, self-cleaving 2A peptide; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. (b) Representative ultrasound images of left renal parenchymal injections in adult mice. White arrow shows kidney location, arrow tip points to the injection needle. (c) Whole body bioluminescence of kidney-specific luciferase expression in representative C57/BL6J mice that underwent ultrasound-guided left intrarenal injection of either ELS or control (no luciferase) lentiviral vector (left hand mouse) at 7 days post injection. The relative luminescence intensity is indicated by a colour scale bar. (d) Representative immunofluorescent images of renal sections of adult (top panels) and neonatal (bottom panels) ELS injected and control (uninjected) kidneys, 60 days post infection. Strawberry expression was limited to the renal cortex and corticomedullary junction. Cor, cortex; Med, medulla. Scale bars, 50 μm.

Mentions: To determine whether lentiviral vectors could be safely and efficiently delivered using a non-surgical approach we utilised a reporter third generation vector in which the expression of Luciferase and Strawberry fluorescent protein is driven by the constitutive EF1a promoter (ELS lentiviral vector, Fig. 1a). We performed single, low volume (10 μl) ultrasound (US) guided microinjection into the left renal parenchyma of adult (8 week, n = 5) and neonatal (7-12 day, n = 5) C57BL/6 mice (Fig. 1b). Each procedure lasted a maximum of 15 minutes and all injected mice recovered well post anaesthesia with no adverse events observed. Histopathological examination of both adult and neonatal kidneys at 7, 15 and 60 days post transduction did not reveal any morphological alterations or infiltrations within the renal parenchyma suggesting the absence of a persistent inflammatory response secondary to lentiviral infection.


A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

Espana-Agusti J, Tuveson DA, Adams DJ, Matakidou A - Sci Rep (2015)

Efficient and sustained renal tubular gene delivery via Ultrasound-guided intraparenchymal injection of lentiviral vectors.(a) Diagram of pELS lentiviral construct. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine tract; EF1, elongation factor 1 alpha promoter; E2A, self-cleaving 2A peptide; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. (b) Representative ultrasound images of left renal parenchymal injections in adult mice. White arrow shows kidney location, arrow tip points to the injection needle. (c) Whole body bioluminescence of kidney-specific luciferase expression in representative C57/BL6J mice that underwent ultrasound-guided left intrarenal injection of either ELS or control (no luciferase) lentiviral vector (left hand mouse) at 7 days post injection. The relative luminescence intensity is indicated by a colour scale bar. (d) Representative immunofluorescent images of renal sections of adult (top panels) and neonatal (bottom panels) ELS injected and control (uninjected) kidneys, 60 days post infection. Strawberry expression was limited to the renal cortex and corticomedullary junction. Cor, cortex; Med, medulla. Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4457145&req=5

f1: Efficient and sustained renal tubular gene delivery via Ultrasound-guided intraparenchymal injection of lentiviral vectors.(a) Diagram of pELS lentiviral construct. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine tract; EF1, elongation factor 1 alpha promoter; E2A, self-cleaving 2A peptide; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. (b) Representative ultrasound images of left renal parenchymal injections in adult mice. White arrow shows kidney location, arrow tip points to the injection needle. (c) Whole body bioluminescence of kidney-specific luciferase expression in representative C57/BL6J mice that underwent ultrasound-guided left intrarenal injection of either ELS or control (no luciferase) lentiviral vector (left hand mouse) at 7 days post injection. The relative luminescence intensity is indicated by a colour scale bar. (d) Representative immunofluorescent images of renal sections of adult (top panels) and neonatal (bottom panels) ELS injected and control (uninjected) kidneys, 60 days post infection. Strawberry expression was limited to the renal cortex and corticomedullary junction. Cor, cortex; Med, medulla. Scale bars, 50 μm.
Mentions: To determine whether lentiviral vectors could be safely and efficiently delivered using a non-surgical approach we utilised a reporter third generation vector in which the expression of Luciferase and Strawberry fluorescent protein is driven by the constitutive EF1a promoter (ELS lentiviral vector, Fig. 1a). We performed single, low volume (10 μl) ultrasound (US) guided microinjection into the left renal parenchyma of adult (8 week, n = 5) and neonatal (7-12 day, n = 5) C57BL/6 mice (Fig. 1b). Each procedure lasted a maximum of 15 minutes and all injected mice recovered well post anaesthesia with no adverse events observed. Histopathological examination of both adult and neonatal kidneys at 7, 15 and 60 days post transduction did not reveal any morphological alterations or infiltrations within the renal parenchyma suggesting the absence of a persistent inflammatory response secondary to lentiviral infection.

Bottom Line: The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation.We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection.Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Cambridge, CRUK Cambridge institute, Cambridge, CB2 0RE, UK.

ABSTRACT
The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.

No MeSH data available.


Related in: MedlinePlus