Localization of complement factor H gene expression and protein distribution in the mouse outer retina

Zeljka McBride, Sharon L. Oltjen, Roxana A. Radu, Jason Estep, Anthony T. Nguyen, Qizhi Gong, Leonard M Hjelmeland

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Purpose: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods: Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results: Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions: Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.

Original languageEnglish (US)
Pages (from-to)110-123
Number of pages14
JournalMolecular Vision
Volume21
StatePublished - Feb 5 2015

Fingerprint

Complement Factor H
Retina
Gene Expression
Proteins
Immunohistochemistry
Messenger RNA
Choroid
In Situ Hybridization
Blood Proteins
Western Blotting

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Localization of complement factor H gene expression and protein distribution in the mouse outer retina. / McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi; Hjelmeland, Leonard M.

In: Molecular Vision, Vol. 21, 05.02.2015, p. 110-123.

Research output: Contribution to journalArticle

McBride, Zeljka ; Oltjen, Sharon L. ; Radu, Roxana A. ; Estep, Jason ; Nguyen, Anthony T. ; Gong, Qizhi ; Hjelmeland, Leonard M. / Localization of complement factor H gene expression and protein distribution in the mouse outer retina. In: Molecular Vision. 2015 ; Vol. 21. pp. 110-123.
@article{884c67483dbc473b9d10afcf07d76b93,
title = "Localization of complement factor H gene expression and protein distribution in the mouse outer retina",
abstract = "Purpose: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods: Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results: Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions: Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.",
author = "Zeljka McBride and Oltjen, {Sharon L.} and Radu, {Roxana A.} and Jason Estep and Nguyen, {Anthony T.} and Qizhi Gong and Hjelmeland, {Leonard M}",
year = "2015",
month = "2",
day = "5",
language = "English (US)",
volume = "21",
pages = "110--123",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - Localization of complement factor H gene expression and protein distribution in the mouse outer retina

AU - McBride, Zeljka

AU - Oltjen, Sharon L.

AU - Radu, Roxana A.

AU - Estep, Jason

AU - Nguyen, Anthony T.

AU - Gong, Qizhi

AU - Hjelmeland, Leonard M

PY - 2015/2/5

Y1 - 2015/2/5

N2 - Purpose: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods: Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results: Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions: Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.

AB - Purpose: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods: Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results: Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions: Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.

UR - http://www.scopus.com/inward/record.url?scp=84922387813&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84922387813&partnerID=8YFLogxK

M3 - Article

VL - 21

SP - 110

EP - 123

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -