5. Use of a spectral confocal system affords greater control of
signal acquisition versus a strictly filter-based system, and provides the ability for simultaneous use of an increased number of
fluorophores/dyes due to the improved spectra distinguishing
capability.
6. This is based on our established cell sorting strategy (Fig. 2).
Lasers required may differ if antibodies different than those
listed for flow cytometry and sorting are used. A cell sorter with
the capability of detecting mCherry is required in order to sort
mCherry M. tuberculosis-infected lung phagocytes.
7. If the promoter region of the gene of interest for the reporter
construct has not been experimentally defined, we examine the
genomic location (distance of upstream gene, presence of the
gene in an operon) and the location of transcription start sites
[35] in determining a putative promoter region. Typical promoter lengths used in reporter M. tuberculosis constructs span
500–700 bp. It can be helpful to generate two reporter constructs containing different putative promoter lengths, to test
for GFP signal induction empirically. We do not in general
introduce an additional “canonical” Shine-Dalgarno ribosome
binding site in the reporter constructs.
8. To generate a translational fusion reporter, such as the
SSB-GFP reporter, follow the same protocol as in Subheading
3.1, but with PCR amplification of the native promoter
together with the open reading frame of the gene of interest.
9. The GFP reporter should be cloned such that it is located
upstream of the constitutively expressed smyc’::mCherry, in
the same transcriptional direction. Expression of both fluorescent reporters on the same vector is critical, as it makes certain
that any bacterium that may lose the plasmid during in vivo
infection (where selective pressure is not generally applied) is
neither visualized nor quantified, ensuring robustness of data
analysis.
10. Intranasal infection provides a robust and straightforward
route for inoculation, and we obtain very similar bacterial
loads with this method as compared to infection via an aerosol
route. These fluorescent reporter M. tuberculosis strains can
however also be used for infection via other routes as desired
or needed.
11. The breathing rate of the mice during isoflurane administration
should be carefully monitored, with the ideal time for intranasal administration of the bacterial inoculum being the point at
which the breathing rate of the mouse first slows significantly.
12. It is important that the tissues be promptly removed from the
fixative and transferred to PBS after the overnight fixation step,
as prolonged incubation in fixative may result in increased
background fluorescence.
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David Giacalone et al.
signal acquisition versus a strictly filter-based system, and provides the ability for simultaneous use of an increased number of
fluorophores/dyes due to the improved spectra distinguishing
capability.
6. This is based on our established cell sorting strategy (Fig. 2).
Lasers required may differ if antibodies different than those
listed for flow cytometry and sorting are used. A cell sorter with
the capability of detecting mCherry is required in order to sort
mCherry M. tuberculosis-infected lung phagocytes.
7. If the promoter region of the gene of interest for the reporter
construct has not been experimentally defined, we examine the
genomic location (distance of upstream gene, presence of the
gene in an operon) and the location of transcription start sites
[35] in determining a putative promoter region. Typical promoter lengths used in reporter M. tuberculosis constructs span
500–700 bp. It can be helpful to generate two reporter constructs containing different putative promoter lengths, to test
for GFP signal induction empirically. We do not in general
introduce an additional “canonical” Shine-Dalgarno ribosome
binding site in the reporter constructs.
8. To generate a translational fusion reporter, such as the
SSB-GFP reporter, follow the same protocol as in Subheading
3.1, but with PCR amplification of the native promoter
together with the open reading frame of the gene of interest.
9. The GFP reporter should be cloned such that it is located
upstream of the constitutively expressed smyc’::mCherry, in
the same transcriptional direction. Expression of both fluorescent reporters on the same vector is critical, as it makes certain
that any bacterium that may lose the plasmid during in vivo
infection (where selective pressure is not generally applied) is
neither visualized nor quantified, ensuring robustness of data
analysis.
10. Intranasal infection provides a robust and straightforward
route for inoculation, and we obtain very similar bacterial
loads with this method as compared to infection via an aerosol
route. These fluorescent reporter M. tuberculosis strains can
however also be used for infection via other routes as desired
or needed.
11. The breathing rate of the mice during isoflurane administration
should be carefully monitored, with the ideal time for intranasal administration of the bacterial inoculum being the point at
which the breathing rate of the mouse first slows significantly.
12. It is important that the tissues be promptly removed from the
fixative and transferred to PBS after the overnight fixation step,
as prolonged incubation in fixative may result in increased
background fluorescence.
380
David Giacalone et al.