In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils

Jacqueline U McDonald, Andrea Cortini, Marcela Rosas, Liliane Fossati-Jimack, Guang Sheng Ling, Kimberley J Lewis, Sharon Dewitt, Kate Liddiard, Gordon D Brown, Simon A Jones, Maurice B Hallett, Marina Botto, Philip R Taylor

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.-McDonald, J. U., Cortini, A., Rosas, M., Fossati-Jimack, L., Ling, G. S., Lewis, K. J., Dewitt, S., Liddiard, K., Brown, G. D., Jones, S. A., Hallett, M. B., Botto, M., Taylor, P. R. In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.
Original languageEnglish
Pages (from-to)2-11
Number of pages11
JournalThe FASEB Journal
Volume25
DOIs
Publication statusPublished - 2011

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Functional analysis
Animals
Neutrophils
Genes
Cells
Throughput
In Vitro Techniques
Single-Cell Analysis
Genetically Modified Animals

Keywords

  • Inflammation
  • Animal models
  • Conditional imortalization

Cite this

McDonald, J. U., Cortini, A., Rosas, M., Fossati-Jimack, L., Ling, G. S., Lewis, K. J., ... Taylor, P. R. (2011). In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils. The FASEB Journal, 25, 2-11. https://doi.org/10.1096/fj.10-178517

In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils. / McDonald, Jacqueline U; Cortini, Andrea; Rosas, Marcela; Fossati-Jimack, Liliane; Ling, Guang Sheng; Lewis, Kimberley J; Dewitt, Sharon; Liddiard, Kate; Brown, Gordon D; Jones, Simon A; Hallett, Maurice B; Botto, Marina; Taylor, Philip R.

In: The FASEB Journal, Vol. 25, 2011, p. 2-11.

Research output: Contribution to journalArticle

McDonald, JU, Cortini, A, Rosas, M, Fossati-Jimack, L, Ling, GS, Lewis, KJ, Dewitt, S, Liddiard, K, Brown, GD, Jones, SA, Hallett, MB, Botto, M & Taylor, PR 2011, 'In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils', The FASEB Journal, vol. 25, pp. 2-11. https://doi.org/10.1096/fj.10-178517
McDonald, Jacqueline U ; Cortini, Andrea ; Rosas, Marcela ; Fossati-Jimack, Liliane ; Ling, Guang Sheng ; Lewis, Kimberley J ; Dewitt, Sharon ; Liddiard, Kate ; Brown, Gordon D ; Jones, Simon A ; Hallett, Maurice B ; Botto, Marina ; Taylor, Philip R. / In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils. In: The FASEB Journal. 2011 ; Vol. 25. pp. 2-11.
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AU - Cortini, Andrea

AU - Rosas, Marcela

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AU - Ling, Guang Sheng

AU - Lewis, Kimberley J

AU - Dewitt, Sharon

AU - Liddiard, Kate

AU - Brown, Gordon D

AU - Jones, Simon A

AU - Hallett, Maurice B

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AU - Taylor, Philip R

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N2 - Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.-McDonald, J. U., Cortini, A., Rosas, M., Fossati-Jimack, L., Ling, G. S., Lewis, K. J., Dewitt, S., Liddiard, K., Brown, G. D., Jones, S. A., Hallett, M. B., Botto, M., Taylor, P. R. In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.

AB - Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.-McDonald, J. U., Cortini, A., Rosas, M., Fossati-Jimack, L., Ling, G. S., Lewis, K. J., Dewitt, S., Liddiard, K., Brown, G. D., Jones, S. A., Hallett, M. B., Botto, M., Taylor, P. R. In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.

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