Abstract
The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 430-442 |
| Number of pages | 13 |
| Journal | Cytometry Part A |
| Volume | 89 |
| Issue number | 5 |
| DOIs | |
| State | Published - May 1 2016 |
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Keywords
- development
- flow cytometry
- gene expression
- oncogenesis
- sorting
ASJC Scopus subject areas
- Pathology and Forensic Medicine
- Histology
- Cell Biology
Cite this
Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states. / Samadder, Partha; Weng, Ning; Doetschman, Thomas C; Heimark, Ronald L; Galbraith, David W.
In: Cytometry Part A, Vol. 89, No. 5, 01.05.2016, p. 430-442.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states
AU - Samadder, Partha
AU - Weng, Ning
AU - Doetschman, Thomas C
AU - Heimark, Ronald L
AU - Galbraith, David W
PY - 2016/5/1
Y1 - 2016/5/1
N2 - The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis.
AB - The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis.
KW - development
KW - flow cytometry
KW - gene expression
KW - oncogenesis
KW - sorting
UR - http://www.scopus.com/inward/record.url?scp=84977621006&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84977621006&partnerID=8YFLogxK
U2 - 10.1002/cyto.a.22847
DO - 10.1002/cyto.a.22847
M3 - Article
C2 - 27003621
AN - SCOPUS:84977621006
VL - 89
SP - 430
EP - 442
JO - Cytometry. Part A : the journal of the International Society for Analytical Cytology
JF - Cytometry. Part A : the journal of the International Society for Analytical Cytology
SN - 1552-4922
IS - 5
ER -