2.1.3 gRNA Libraries
An alternative way of achieving sequence/tissue-specific gene disruption is to use the CrispR/Cas9 system. This approach uses a
guide RNA (gRNA) construct for a gene of interest, which is
ubiquitously expressed under the control of an RNApol III promoter. When this is combined with a GAL4 driver and UAS-Cas9,
one can achieve tissue-specific somatic cutting of the gene of interest [33]. Genome-wide gRNA libraries are currently being created
as part of the Harvard Medical School Transgenic RNAi Project
(TRiP), available from the BDSC, and by NIG-FLY. UAS-Cas9
stocks are available at both the BDSC and NIG-FLY stock centers.
2.1.4 Fluorescent
Reporter Stocks for EMT
Factors
Analysis of the changes undergone by PE cells undergoing pEMT is
facilitated by fluorescent markers for cellular and extracellular molecules that play important roles in EMT. Listed below (Table 3) are a
useful set of such fly stocks which label molecules associated with
the basement membrane (BM), adherens junctions (AJs), F-Actin,
and myosin. While analysis of cellular and extracellular changes is
straightforward with ex vivo cultured discs, obtaining clear visualization of the pEMT event as it occurs in vivo is challenging, due to
the complex environment. For in vivo analysis, transgenic fluorescent reporters are useful as their expression can be restricted to
peripodial cells using genetic means (i.e., GAL4 drivers or direct
promoter fusions) to make them stand out from the surrounding
cells [13] and they can remove the need for immunostaining. We
have listed some fluorescent UAS lines here that are useful, such as
the F-Actin reporters, but many other UAS fluorophore stocks are
available at the stock centers. We have also included reporters for
the JNK pathway which plays a central role in wing disc eversion,
and the netrin receptor Frazzled which our lab has identified as an
inhibitor of the EMT event [21]. In addition, there are several
other genes and pathways that are not listed here, which have
been shown to play a role in eversion such as Pvr [18] and Dpp
[20]. Some care should be taken when using fluorescently tagged
genes to visualize key mediators of EMT such as E-Cad and F-Actin
as the use of non-endogenous regulation (e.g., ubiquitous promoters, GAL4/UAS) and the tag itself can alter the normal levels of
expression, sub-cellular localization, and stability/dynamics (see
Notes 1 and 2).
2.2 Solutions
1. Shields and Sang M3 insect medium.
2. Penicillin-Streptomycin.
3. Fetal calf serum.
4. 20-Hydroxyecdysone.
5. Triton X-100.
6. Glycerol.
7. Oil for adhering pupa to glass (e.g., Liquid paraffin or
voltalef 10S).
120
Sofia Golenkina et al.
An alternative way of achieving sequence/tissue-specific gene disruption is to use the CrispR/Cas9 system. This approach uses a
guide RNA (gRNA) construct for a gene of interest, which is
ubiquitously expressed under the control of an RNApol III promoter. When this is combined with a GAL4 driver and UAS-Cas9,
one can achieve tissue-specific somatic cutting of the gene of interest [33]. Genome-wide gRNA libraries are currently being created
as part of the Harvard Medical School Transgenic RNAi Project
(TRiP), available from the BDSC, and by NIG-FLY. UAS-Cas9
stocks are available at both the BDSC and NIG-FLY stock centers.
2.1.4 Fluorescent
Reporter Stocks for EMT
Factors
Analysis of the changes undergone by PE cells undergoing pEMT is
facilitated by fluorescent markers for cellular and extracellular molecules that play important roles in EMT. Listed below (Table 3) are a
useful set of such fly stocks which label molecules associated with
the basement membrane (BM), adherens junctions (AJs), F-Actin,
and myosin. While analysis of cellular and extracellular changes is
straightforward with ex vivo cultured discs, obtaining clear visualization of the pEMT event as it occurs in vivo is challenging, due to
the complex environment. For in vivo analysis, transgenic fluorescent reporters are useful as their expression can be restricted to
peripodial cells using genetic means (i.e., GAL4 drivers or direct
promoter fusions) to make them stand out from the surrounding
cells [13] and they can remove the need for immunostaining. We
have listed some fluorescent UAS lines here that are useful, such as
the F-Actin reporters, but many other UAS fluorophore stocks are
available at the stock centers. We have also included reporters for
the JNK pathway which plays a central role in wing disc eversion,
and the netrin receptor Frazzled which our lab has identified as an
inhibitor of the EMT event [21]. In addition, there are several
other genes and pathways that are not listed here, which have
been shown to play a role in eversion such as Pvr [18] and Dpp
[20]. Some care should be taken when using fluorescently tagged
genes to visualize key mediators of EMT such as E-Cad and F-Actin
as the use of non-endogenous regulation (e.g., ubiquitous promoters, GAL4/UAS) and the tag itself can alter the normal levels of
expression, sub-cellular localization, and stability/dynamics (see
Notes 1 and 2).
2.2 Solutions
1. Shields and Sang M3 insect medium.
2. Penicillin-Streptomycin.
3. Fetal calf serum.
4. 20-Hydroxyecdysone.
5. Triton X-100.
6. Glycerol.
7. Oil for adhering pupa to glass (e.g., Liquid paraffin or
voltalef 10S).
120
Sofia Golenkina et al.