Abstract
Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any ftuorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.
| Original language | English |
|---|---|
| Journal | American Journal of Physiology - Renal Physiology |
| Volume | 289 |
| Issue number | 3 58-3 |
| DOIs | |
| State | Published - Sep 2005 |
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Keywords
- Actin
- Actin depolymerizing factor
- Adenovirus
- Green fluorescent protein
- Intravital microscopy
ASJC Scopus subject areas
- Physiology
Cite this
Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney. / Tanner, George A.; Sandoval, Ruben M.; Molitoris, Bruce; Bamburg, James R.; Ashworth, Sharon L.
In: American Journal of Physiology - Renal Physiology, Vol. 289, No. 3 58-3, 09.2005.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney
AU - Tanner, George A.
AU - Sandoval, Ruben M.
AU - Molitoris, Bruce
AU - Bamburg, James R.
AU - Ashworth, Sharon L.
PY - 2005/9
Y1 - 2005/9
N2 - Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any ftuorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.
AB - Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any ftuorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.
KW - Actin
KW - Actin depolymerizing factor
KW - Adenovirus
KW - Green fluorescent protein
KW - Intravital microscopy
UR - http://www.scopus.com/inward/record.url?scp=23944445770&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=23944445770&partnerID=8YFLogxK
U2 - 10.1152/ajprenal.00059.2005
DO - 10.1152/ajprenal.00059.2005
M3 - Article
VL - 289
JO - American Journal of Physiology
JF - American Journal of Physiology
SN - 0193-1857
IS - 3 58-3
ER -