immersion objective. Cells can be counter-stained with
Hoechst (0.1 μg/mL) to observe subcellular localization of
the fusion protein.
3. For imaging of fixed cells, wash the cells with PBS, fix them
with 4% PFA in PBS and again twice with PBS. Record images
with a 63Â/1.4 NA immersion objective. Although fixed cells
can be stored for a few weeks at 4
C, try to observe them
1–2 days after fixation. Cells can be counter-stained with antibodies to confirm expression of the target protein or for
co-localization experiments.
A live cell image of U2-OS
::XPB-GFP cells in which the XPB gene
is fused to eGFP is shown in Fig. 6b.
4 Notes
1. Detailed information is available on the ATCC website
(https://www.lgcstandards-atcc.org/). A guide containing
general technical information for working with animal cells in
culture, including media, sub-culturing, cryopreservation, and
thawing is also accessible.
2. Affinity tags can be introduced at the N- or C- end of the target
protein or inserted into internal loops. It is wise to perform
extensive literature search for functionality studies and expression data in human cells as well as in other species such as
mouse, C. elegans, or yeast to avoid trying to tag an extremity
that would compromise protein function. In the absence of
information on the accessibility of the extremities of the target
protein, a C-terminal tag is generally preferred [28].
3. Nucleofection relies on permeabilization of the cellular membranes by electroporation. Although Amaxa nucleofection is
often superior to other widely used transfection approaches,
lipid-based transfection with reagents such as Lipofectamine
3000™ or jet-CRISPR™ can also be used.
4. Homology-based cloning techniques including sequence and
ligation–independent cloning (SLIC) and commercially available technologies such as In-Fusion and Gibson assembly are
perfectly adapted to assemble three to four DNA fragments in a
single step reaction. These technologies are now routinely used
in many laboratories (see ref. [29] in this issue) and can be used
to rapidly generate the donor plasmid. The homology arms can
be amplified from genomic DNA and the insert from plasmids
which can be obtained from non-profit organizations (e.g., see
Addgene plasmids #112848, #52379). Note that a growing
number of companies also offer donor plasmid design and
preparation as a service.
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
55
Hoechst (0.1 μg/mL) to observe subcellular localization of
the fusion protein.
3. For imaging of fixed cells, wash the cells with PBS, fix them
with 4% PFA in PBS and again twice with PBS. Record images
with a 63Â/1.4 NA immersion objective. Although fixed cells
can be stored for a few weeks at 4
C, try to observe them
1–2 days after fixation. Cells can be counter-stained with antibodies to confirm expression of the target protein or for
co-localization experiments.
A live cell image of U2-OS
::XPB-GFP cells in which the XPB gene
is fused to eGFP is shown in Fig. 6b.
4 Notes
1. Detailed information is available on the ATCC website
(https://www.lgcstandards-atcc.org/). A guide containing
general technical information for working with animal cells in
culture, including media, sub-culturing, cryopreservation, and
thawing is also accessible.
2. Affinity tags can be introduced at the N- or C- end of the target
protein or inserted into internal loops. It is wise to perform
extensive literature search for functionality studies and expression data in human cells as well as in other species such as
mouse, C. elegans, or yeast to avoid trying to tag an extremity
that would compromise protein function. In the absence of
information on the accessibility of the extremities of the target
protein, a C-terminal tag is generally preferred [28].
3. Nucleofection relies on permeabilization of the cellular membranes by electroporation. Although Amaxa nucleofection is
often superior to other widely used transfection approaches,
lipid-based transfection with reagents such as Lipofectamine
3000™ or jet-CRISPR™ can also be used.
4. Homology-based cloning techniques including sequence and
ligation–independent cloning (SLIC) and commercially available technologies such as In-Fusion and Gibson assembly are
perfectly adapted to assemble three to four DNA fragments in a
single step reaction. These technologies are now routinely used
in many laboratories (see ref. [29] in this issue) and can be used
to rapidly generate the donor plasmid. The homology arms can
be amplified from genomic DNA and the insert from plasmids
which can be obtained from non-profit organizations (e.g., see
Addgene plasmids #112848, #52379). Note that a growing
number of companies also offer donor plasmid design and
preparation as a service.
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
55