In vitro transfection of cultured human retinal vascular endothelial cells (HREC) with a ribozyme (Rz) targeted against Insulin-like Growth Factor (IGF-I) receptor (Rc) mRNA

S. Caballero, R. W. Tarnuzzer, D. M. Bush, M. B. Grant

Research output: Contribution to journalArticle

Abstract

Purpose. IGF-I is implicated in diabetic retinopathy (DR). Its effects are mediated by a specific cell surface Rc. By blocking expression of IGF-I Rc in cultured HREC we can further elucidate the contribution of this growth factor to aberrant HREC function in DR. In order to accomplish this we chose to transfert cells in vitro with a synthetic catalytic RNA molecule (ribozyme) targeted against IGF-I Rc mRNA. Methods. We used consensus sequence for the "hammerhead" type Rz to search GenBank for catalytic sites within human IGF-I type 1 Rc mRNA. We then designed and synthesized a 39 nucleotide (nt) Rz with specificity to this message. We also designed and synthesized a radiolabelled 16 nt RNA target to test the activity and specificity of the Rz in a cell-free system. Reaction products were analyzed by auto-radiography after electrophoretic separation. To optimize transfection conditions using cationic liposomes we then synthesized a DNA analogue of the Rz. This Rz analogue was labelled with digoxigenin (dig) prior to packaging in one of three commercial liposome preparations. Enzyme-conjugated anti-dig with appropriate substrate was used to measure the degree of transfecton in HREC. After testing the Rz in the cell-free system and determining the optimum transfection conditions, we used competition-based quantitative RT-PCR to examine the tevel of IGF-I Rc mRNA in control and Rz-transfected cultured HREC. Results. In the cell-free system, IGF-I Rc Rz specifically cleaved radiolabeled synthetic target RNA sequence, as evidenced by the appearance of 9 nt and 7 nt bands. Of three commercial cationic liposome preparations tested, DOTAP reulted in the lowest cell mortality and gave the greatest staining intensity for dig-labelled oligo within the cells. When Rz was transfected into cultured HREC, we could measure no change in IGF-I Rc mRNA after 2 hr exposure to Rz. Conclusion. We succesfully demonstrated Rz catalytic activity in a cell-free system using a Rz targeted against IGF-I Rc. We also determined that oligonucleotides can be delivered into HREC using cationic liposome-mediated transfection. The inability to detect Rz activity in transfected cells by a drop in IGF-I Rc mRNA levels could have several causes. Longer times of incubation post transfection may be required to defect a measurable decrease in target mRNA. Alternatively, the Rz could be degraded by endogenous RNAses prior to their having a measurable effect. We are currently synthesizing chemically modified Rz which are "protected" from RNAse degradation. Finally, Rz coding sequence could be cloned into an expression vector, which would then be transfected into cells for continuous Rz expression as a way of overcoming rapid degradation. Ribozymes can be a powerful tool to examine the effects of specific effectors on cultured cells by blocking synthesis of receptors for those molecules at the mRNA level, and can therefore help our understanding of the processes involved in DR.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996
Externally publishedYes

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Retinal Vessels
Catalytic RNA
IGF Type 1 Receptor
Transfection
Endothelial Cells
Messenger RNA
Cell-Free System
Digoxigenin
Liposomes
Diabetic Retinopathy
Nucleotides
In Vitro Techniques

ASJC Scopus subject areas

  • Ophthalmology

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In vitro transfection of cultured human retinal vascular endothelial cells (HREC) with a ribozyme (Rz) targeted against Insulin-like Growth Factor (IGF-I) receptor (Rc) mRNA. / Caballero, S.; Tarnuzzer, R. W.; Bush, D. M.; Grant, M. B.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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title = "In vitro transfection of cultured human retinal vascular endothelial cells (HREC) with a ribozyme (Rz) targeted against Insulin-like Growth Factor (IGF-I) receptor (Rc) mRNA",
abstract = "Purpose. IGF-I is implicated in diabetic retinopathy (DR). Its effects are mediated by a specific cell surface Rc. By blocking expression of IGF-I Rc in cultured HREC we can further elucidate the contribution of this growth factor to aberrant HREC function in DR. In order to accomplish this we chose to transfert cells in vitro with a synthetic catalytic RNA molecule (ribozyme) targeted against IGF-I Rc mRNA. Methods. We used consensus sequence for the {"}hammerhead{"} type Rz to search GenBank for catalytic sites within human IGF-I type 1 Rc mRNA. We then designed and synthesized a 39 nucleotide (nt) Rz with specificity to this message. We also designed and synthesized a radiolabelled 16 nt RNA target to test the activity and specificity of the Rz in a cell-free system. Reaction products were analyzed by auto-radiography after electrophoretic separation. To optimize transfection conditions using cationic liposomes we then synthesized a DNA analogue of the Rz. This Rz analogue was labelled with digoxigenin (dig) prior to packaging in one of three commercial liposome preparations. Enzyme-conjugated anti-dig with appropriate substrate was used to measure the degree of transfecton in HREC. After testing the Rz in the cell-free system and determining the optimum transfection conditions, we used competition-based quantitative RT-PCR to examine the tevel of IGF-I Rc mRNA in control and Rz-transfected cultured HREC. Results. In the cell-free system, IGF-I Rc Rz specifically cleaved radiolabeled synthetic target RNA sequence, as evidenced by the appearance of 9 nt and 7 nt bands. Of three commercial cationic liposome preparations tested, DOTAP reulted in the lowest cell mortality and gave the greatest staining intensity for dig-labelled oligo within the cells. When Rz was transfected into cultured HREC, we could measure no change in IGF-I Rc mRNA after 2 hr exposure to Rz. Conclusion. We succesfully demonstrated Rz catalytic activity in a cell-free system using a Rz targeted against IGF-I Rc. We also determined that oligonucleotides can be delivered into HREC using cationic liposome-mediated transfection. The inability to detect Rz activity in transfected cells by a drop in IGF-I Rc mRNA levels could have several causes. Longer times of incubation post transfection may be required to defect a measurable decrease in target mRNA. Alternatively, the Rz could be degraded by endogenous RNAses prior to their having a measurable effect. We are currently synthesizing chemically modified Rz which are {"}protected{"} from RNAse degradation. Finally, Rz coding sequence could be cloned into an expression vector, which would then be transfected into cells for continuous Rz expression as a way of overcoming rapid degradation. Ribozymes can be a powerful tool to examine the effects of specific effectors on cultured cells by blocking synthesis of receptors for those molecules at the mRNA level, and can therefore help our understanding of the processes involved in DR.",
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T1 - In vitro transfection of cultured human retinal vascular endothelial cells (HREC) with a ribozyme (Rz) targeted against Insulin-like Growth Factor (IGF-I) receptor (Rc) mRNA

AU - Caballero, S.

AU - Tarnuzzer, R. W.

AU - Bush, D. M.

AU - Grant, M. B.

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N2 - Purpose. IGF-I is implicated in diabetic retinopathy (DR). Its effects are mediated by a specific cell surface Rc. By blocking expression of IGF-I Rc in cultured HREC we can further elucidate the contribution of this growth factor to aberrant HREC function in DR. In order to accomplish this we chose to transfert cells in vitro with a synthetic catalytic RNA molecule (ribozyme) targeted against IGF-I Rc mRNA. Methods. We used consensus sequence for the "hammerhead" type Rz to search GenBank for catalytic sites within human IGF-I type 1 Rc mRNA. We then designed and synthesized a 39 nucleotide (nt) Rz with specificity to this message. We also designed and synthesized a radiolabelled 16 nt RNA target to test the activity and specificity of the Rz in a cell-free system. Reaction products were analyzed by auto-radiography after electrophoretic separation. To optimize transfection conditions using cationic liposomes we then synthesized a DNA analogue of the Rz. This Rz analogue was labelled with digoxigenin (dig) prior to packaging in one of three commercial liposome preparations. Enzyme-conjugated anti-dig with appropriate substrate was used to measure the degree of transfecton in HREC. After testing the Rz in the cell-free system and determining the optimum transfection conditions, we used competition-based quantitative RT-PCR to examine the tevel of IGF-I Rc mRNA in control and Rz-transfected cultured HREC. Results. In the cell-free system, IGF-I Rc Rz specifically cleaved radiolabeled synthetic target RNA sequence, as evidenced by the appearance of 9 nt and 7 nt bands. Of three commercial cationic liposome preparations tested, DOTAP reulted in the lowest cell mortality and gave the greatest staining intensity for dig-labelled oligo within the cells. When Rz was transfected into cultured HREC, we could measure no change in IGF-I Rc mRNA after 2 hr exposure to Rz. Conclusion. We succesfully demonstrated Rz catalytic activity in a cell-free system using a Rz targeted against IGF-I Rc. We also determined that oligonucleotides can be delivered into HREC using cationic liposome-mediated transfection. The inability to detect Rz activity in transfected cells by a drop in IGF-I Rc mRNA levels could have several causes. Longer times of incubation post transfection may be required to defect a measurable decrease in target mRNA. Alternatively, the Rz could be degraded by endogenous RNAses prior to their having a measurable effect. We are currently synthesizing chemically modified Rz which are "protected" from RNAse degradation. Finally, Rz coding sequence could be cloned into an expression vector, which would then be transfected into cells for continuous Rz expression as a way of overcoming rapid degradation. Ribozymes can be a powerful tool to examine the effects of specific effectors on cultured cells by blocking synthesis of receptors for those molecules at the mRNA level, and can therefore help our understanding of the processes involved in DR.

AB - Purpose. IGF-I is implicated in diabetic retinopathy (DR). Its effects are mediated by a specific cell surface Rc. By blocking expression of IGF-I Rc in cultured HREC we can further elucidate the contribution of this growth factor to aberrant HREC function in DR. In order to accomplish this we chose to transfert cells in vitro with a synthetic catalytic RNA molecule (ribozyme) targeted against IGF-I Rc mRNA. Methods. We used consensus sequence for the "hammerhead" type Rz to search GenBank for catalytic sites within human IGF-I type 1 Rc mRNA. We then designed and synthesized a 39 nucleotide (nt) Rz with specificity to this message. We also designed and synthesized a radiolabelled 16 nt RNA target to test the activity and specificity of the Rz in a cell-free system. Reaction products were analyzed by auto-radiography after electrophoretic separation. To optimize transfection conditions using cationic liposomes we then synthesized a DNA analogue of the Rz. This Rz analogue was labelled with digoxigenin (dig) prior to packaging in one of three commercial liposome preparations. Enzyme-conjugated anti-dig with appropriate substrate was used to measure the degree of transfecton in HREC. After testing the Rz in the cell-free system and determining the optimum transfection conditions, we used competition-based quantitative RT-PCR to examine the tevel of IGF-I Rc mRNA in control and Rz-transfected cultured HREC. Results. In the cell-free system, IGF-I Rc Rz specifically cleaved radiolabeled synthetic target RNA sequence, as evidenced by the appearance of 9 nt and 7 nt bands. Of three commercial cationic liposome preparations tested, DOTAP reulted in the lowest cell mortality and gave the greatest staining intensity for dig-labelled oligo within the cells. When Rz was transfected into cultured HREC, we could measure no change in IGF-I Rc mRNA after 2 hr exposure to Rz. Conclusion. We succesfully demonstrated Rz catalytic activity in a cell-free system using a Rz targeted against IGF-I Rc. We also determined that oligonucleotides can be delivered into HREC using cationic liposome-mediated transfection. The inability to detect Rz activity in transfected cells by a drop in IGF-I Rc mRNA levels could have several causes. Longer times of incubation post transfection may be required to defect a measurable decrease in target mRNA. Alternatively, the Rz could be degraded by endogenous RNAses prior to their having a measurable effect. We are currently synthesizing chemically modified Rz which are "protected" from RNAse degradation. Finally, Rz coding sequence could be cloned into an expression vector, which would then be transfected into cells for continuous Rz expression as a way of overcoming rapid degradation. Ribozymes can be a powerful tool to examine the effects of specific effectors on cultured cells by blocking synthesis of receptors for those molecules at the mRNA level, and can therefore help our understanding of the processes involved in DR.

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