3. Localization errors are estimated from the distributions of the
distance between the co-localized spots measured from fluorescent beads and live embryo control constructs. The variance
of distance distribution from these controls can be subtracted
from the mean squared distances between two spots obtained
from a time series of measurement (e.g., Fig. 2d, e).
3.6 Image Analysis
Custom MATLAB code is available upon request. The code
includes nuclear registration, spot segmentation and tracking,
chromatic aberration correction, and MSD analysis. Detailed
descriptions can be found in [18].
4 Notes
1. In these lines, Cas9 is materially expressed from a nos-Cas9 or
vas-Cas9 source integrated at designated genomic locations.
See genome editing resource at Bloomington Drosophila Stock
Center (BDSC) for more information (https://bdsc.indiana.
edu/stocks/genome_editing/crispr_cas9.html). In this protocol, we engineered the second chromosome as an example and
used BDSC #51324, in which a vas-Cas9 transgene is inserted
at the third chromosome and marked with a body color marker
(yellow, y).
2. We used BDSC #34770 as the maternal phiC31 integrase
source. These female flies provide integrase activity in their
germlines, which induces attB/attP recombination to incorporate transgenes. For more information about the selection of
integrase lines, see https://bdsc.indiana.edu/stocks/phic31/
index.html.
3. eGFP is fused to the N-terminus of the Burkholderia parB
protein. Due to the slow turnover of ParB proteins at the
parS site, the highly photostable eGFP fusion ensures longtime visualization of the parS-tagged loci. MCP is fused with a
3xtagBFP2. A nuclear localization signal (NLS) is added to the
fusion, which leads to strong nuclear enrichment of the fusion
protein. This increased nuclear background helps to overcome
the relatively low photostability of blue fluorescent proteins
and allows continuous detection of transcription spots across
the blastoderm stage. PCP is fused with 3xmKate2 to provide
good spectral separation from the green channel. An NLS is
added to increase nuclear concentration of the fusion protein.
NLS-PCP-3xmKate2 is enriched in the cytoplasm until 10 min
before gastrulation. Three transgenic lines (nos-NLS-MCP3xtagBFP2, nos-NLS-PCP-3xmKate2, and vas-ParB-eGFP)
were generated separately through germline injections. A
homozygously combined stock (PCP-3xmKate2; vas-ParBeGFP) is maintained and available upon request.
Imaging of Chromatin Loci and Transcription
381
distance between the co-localized spots measured from fluorescent beads and live embryo control constructs. The variance
of distance distribution from these controls can be subtracted
from the mean squared distances between two spots obtained
from a time series of measurement (e.g., Fig. 2d, e).
3.6 Image Analysis
Custom MATLAB code is available upon request. The code
includes nuclear registration, spot segmentation and tracking,
chromatic aberration correction, and MSD analysis. Detailed
descriptions can be found in [18].
4 Notes
1. In these lines, Cas9 is materially expressed from a nos-Cas9 or
vas-Cas9 source integrated at designated genomic locations.
See genome editing resource at Bloomington Drosophila Stock
Center (BDSC) for more information (https://bdsc.indiana.
edu/stocks/genome_editing/crispr_cas9.html). In this protocol, we engineered the second chromosome as an example and
used BDSC #51324, in which a vas-Cas9 transgene is inserted
at the third chromosome and marked with a body color marker
(yellow, y).
2. We used BDSC #34770 as the maternal phiC31 integrase
source. These female flies provide integrase activity in their
germlines, which induces attB/attP recombination to incorporate transgenes. For more information about the selection of
integrase lines, see https://bdsc.indiana.edu/stocks/phic31/
index.html.
3. eGFP is fused to the N-terminus of the Burkholderia parB
protein. Due to the slow turnover of ParB proteins at the
parS site, the highly photostable eGFP fusion ensures longtime visualization of the parS-tagged loci. MCP is fused with a
3xtagBFP2. A nuclear localization signal (NLS) is added to the
fusion, which leads to strong nuclear enrichment of the fusion
protein. This increased nuclear background helps to overcome
the relatively low photostability of blue fluorescent proteins
and allows continuous detection of transcription spots across
the blastoderm stage. PCP is fused with 3xmKate2 to provide
good spectral separation from the green channel. An NLS is
added to increase nuclear concentration of the fusion protein.
NLS-PCP-3xmKate2 is enriched in the cytoplasm until 10 min
before gastrulation. Three transgenic lines (nos-NLS-MCP3xtagBFP2, nos-NLS-PCP-3xmKate2, and vas-ParB-eGFP)
were generated separately through germline injections. A
homozygously combined stock (PCP-3xmKate2; vas-ParBeGFP) is maintained and available upon request.
Imaging of Chromatin Loci and Transcription
381