9. Aluminum foil may be used to make this mold with a dimension of 20 mm by 20 mm by 10 mm.
10. Laminin coating may be optional depending on the cell type
seeded. This needs to be optimized. In our preliminary trial, we
found that laminin enhanced cell proliferation of U2OS.EGFP
cells on the scaffolds.
11. Confocal microscopy helps in clearly identifying GFP
+ and
GFP
À cells with just GFP and DAPI signals on the fiber scaffolds. In this case, cytoskeleton staining may not be needed.
Alternatively, other staining or methods that can visualize cellular outline may be used. However, we have tried using WGA
to label cellular membranes, but it did not provide clear cellular
outlines on the fiber scaffolds. Note that the fluorophore chosen shouldn’t be in a similar range of wavelength that the
GFP has.
12. For gene editing evaluation, genomic disruption assay using
U2OS.EGFP is a fast and efficient method. However, for
proper evaluation, genomic cleavage detection (GCD) assay is
also needed to validate the results. Sequencing can also be
performed to check for the indels. If a different scaffold design
or when different reagents are used, loading efficiency and
release profile may be checked through fluorescence tagged
Cas9 or sgRNA. Alternatively, RNA assay (Ribogreen Assay)
can be performed as a proxy for quantifying the sgRNA
amount.
13. The optimal number of scaffolds per microtube depends on the
required number of cells stated on the kit. Here, we used one
scaffold (10, 000 U2OS.EGFP cells were seeded per scaffold
and cultured for 3 days) per sample. If the protocol is modified
or a different cell type is used, the number of scaffolds may
need to be optimized. If more DNA is desired, pool solution
from more scaffolds as one sample.
Acknowledgments
Partial funding support from the Singapore National Research
Foundation under its National Medical Research CouncilCooperative Basic Research Grant (NMRC-CBRG) grant
(NMRC/CBRG/0096/2015) and administered by the Singapore
Ministry of Health’s National Medical Research Council; Ministry
of Education Tier 1 grant (RG38/19); and A*Star BMRC
Singapore-China 12th Joint Research Programme Grant (Project
No: 1610500024) are acknowledged. Jiah Shin Chin would like to
thank the NTU Interdisciplinary Graduate Research Officer’s
scheme for supporting her through this work. We would also like
to thank New England Biolabs for providing the Cas9 proteins.
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Wai Hon Chooi et al.
10. Laminin coating may be optional depending on the cell type
seeded. This needs to be optimized. In our preliminary trial, we
found that laminin enhanced cell proliferation of U2OS.EGFP
cells on the scaffolds.
11. Confocal microscopy helps in clearly identifying GFP
+ and
GFP
À cells with just GFP and DAPI signals on the fiber scaffolds. In this case, cytoskeleton staining may not be needed.
Alternatively, other staining or methods that can visualize cellular outline may be used. However, we have tried using WGA
to label cellular membranes, but it did not provide clear cellular
outlines on the fiber scaffolds. Note that the fluorophore chosen shouldn’t be in a similar range of wavelength that the
GFP has.
12. For gene editing evaluation, genomic disruption assay using
U2OS.EGFP is a fast and efficient method. However, for
proper evaluation, genomic cleavage detection (GCD) assay is
also needed to validate the results. Sequencing can also be
performed to check for the indels. If a different scaffold design
or when different reagents are used, loading efficiency and
release profile may be checked through fluorescence tagged
Cas9 or sgRNA. Alternatively, RNA assay (Ribogreen Assay)
can be performed as a proxy for quantifying the sgRNA
amount.
13. The optimal number of scaffolds per microtube depends on the
required number of cells stated on the kit. Here, we used one
scaffold (10, 000 U2OS.EGFP cells were seeded per scaffold
and cultured for 3 days) per sample. If the protocol is modified
or a different cell type is used, the number of scaffolds may
need to be optimized. If more DNA is desired, pool solution
from more scaffolds as one sample.
Acknowledgments
Partial funding support from the Singapore National Research
Foundation under its National Medical Research CouncilCooperative Basic Research Grant (NMRC-CBRG) grant
(NMRC/CBRG/0096/2015) and administered by the Singapore
Ministry of Health’s National Medical Research Council; Ministry
of Education Tier 1 grant (RG38/19); and A*Star BMRC
Singapore-China 12th Joint Research Programme Grant (Project
No: 1610500024) are acknowledged. Jiah Shin Chin would like to
thank the NTU Interdisciplinary Graduate Research Officer’s
scheme for supporting her through this work. We would also like
to thank New England Biolabs for providing the Cas9 proteins.
190
Wai Hon Chooi et al.