1. shRNA-Specific Regulation of FMNL2
Expression in P19 Cells
Presented By: Gavin West & Yousef Layyous
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2. Introduction - Actin Cytoskeleton Remodelling
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Actin filamentation is important for the formation filopodia
and lamellipodia (Breitsprecher & Goode, 2013).
Formation of these cellular protrusions is very organized
and highly controlled. Regulation involves GTPases, the
Arp ⅔ complex and formin proteins (Grobe,
Wüstenhagen, Baarlink, Grosse, & Grikscheit, 2018).
Understanding mechanisms surrounding actin
cytoskeleton modelling has important applications in
understanding cell differentiation and cancer metastasis
(Grikscheit, Frank, Wang, & Grosse, 2015).
Research gate
(“The different facets of actin cytoskeleton remodeling in migrating
T... | Download Scientific Diagram,” n.d.)

3. Introduction - FMNL2 Function in Actin Remodelling
The Formin-related proteins are the largest group of Rho
GTPase effectors (Grikscheit, Frank, Wang, & Grosse,
2015).
- What are their functions?
Formin-like 2 (FMNL2) is a member of the
diaphanous-related formins (DRFs). FMNL2 has a critical
role in junctional actin assembly in cell-cell contact formation
(Péladeau, Heibein, Maltez, Copeland, & Copeland, 2016).
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4. Introduction - RNAi for Gene Suppression
RNA interference is a regulatory process that
controls post-transcriptional gene silencing.
Vector-based short hairpin RNA (shRNA) is a type
of RNAi useful for studying gene functions by knock
down of cytosolic mRNA (Moore, Guthrie, Huang, &
Taxman, 2010).
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(“shRNA Process and shRNA Diagram | Sigma-Aldrich,” n.d.)

5. Introduction - Research Goal
The goal of this semester's research project was to generate an shRNA specific for formin-like 2 (FMNL2)
and to observe its impact on gene expression.
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6. Result - Which temperature is Better?
Fig 1. PCR products from primer specificity evaluation.
Five different PCR reactions and a negative control were used to study the effect of temperature in PCR amplification as well as the
specificity of the primer; 1% agarose gel was stained with ethidium bromide to visualize the DNA lanes. The temperature for each
PCR is stated on top of each lane. All lanes show a specific product of 330 bp long.
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Ladder
68.9°C
50.0 °C
54.1°C
58.8°C
62.7°C
400bp
300bp
ShRNA

7. Result - Bacterial Transformation with pDrive.
Blue white screening was used to confirm the transformation success of one colony. Ω number of colonies with no insert, which
appears blue in culture; α number of colonies with insert, which appeared white in culture; λ ratio between α/ Ω. λ was calculate by
dividing Ω over α. The bacteria were transformed using recombinant p Drive vector.
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8. Fig 2. Screening pDrive vector for presence of shRNA-expressing construct.
After a dirty digest of 6 white colonies, the vector pDrive was screened for the presence of an shRNA-expressing DNA construct. The digest
products were analyzed by of ethidium bromide stained gel electrophoresis. (A) The results of group W1. (B) Digestion results borrowed from
group W3. First a 10 kb DNA ladder was loaded to the gel, followed by six DNA amplicon digestion experiments isolated from bacterial
colonies. 3 of the DNA amplicons were present in pDrive, bands present at roughly 400 bp.
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Result - Was DNA Construct Successfully Inserted into pDrive?
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Fig 3. Densitometry results of for DNA construct and vector concentration.
Densitometry was used to determine concentration of DNA insert and pEGFP-C3 vector. Insert and vector products were analyzed by ethidium
bromide stained gel electrophoresis. Concentration was measured using AlphaView by comparing band intensity to the ladder. A) The DNA insert
is located on the gel at roughly 400 bp. Vector is not present in panel A as it was not loaded. (B) Results of ethidium bromide stained gel
electrophoresis of ligation of digested DNA construct into pEGFP-C3 vector. Y corresponds to the DNA construct of roughly 400 bp. The vector
and 1/3 diluted vector can be visualized at approximately 5000 bp. Results were borrowed from group W8. The results were quantitatively
measured using the AlphaView, comparing the DNA ladder to the intensity of the bands.
400 Bp
5000 Bp
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Result - Determination of DNA Concentration for Ligation
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DNA Origin DNA Mass (ng)
Insert 7.47
Vector 26.6
Diluted Vector
3X
8.86

10. Result - Bacterial Transformation of pEGFP-C3
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Vector (CFU) Vector + Insert (CFU) Ratio of Vector to Vector+Insert
38 36 1:1
Table 2. Yield of DNA construct insertion into vector during transformation.
Two plates containing TOP 10 E. coli cells were transformed using the pEGFP-C3 vector. Presented in the table are the
colony counts. Ω is a ratio of the vector to vector plus ratio. Being calculated as vector+insert/ vector, ratio was rounded to the
nearest whole number. Cells were plated on agar plates containing kanamycin as a selective plate. The DNA insert was a
specific shRNA for FMNL2.

11. Result - Which colonies were transformed?
Fig 4. PCR screening of transformed cells with vector pEGFP-C3 containing ShRNA construct.
A transformation reaction was made using vector with specific ShRNA against FMNL2. LB Bacterial broth was used to culture six random
colonies from plate where the transformed cells where grown. After PCR, the products where visualized in a 1% agarose gel electrophoresis
stained with ethidium bromide.
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12. μ
Ns
Sp
TL FM
Fig 5. Microscopy examination of P19 transfected cells for EGFP expression.
Two P19 cell cultures where transfected with DNA constructs, one with shRNA specific (Sp) for FMNL2 and a second DNA construct with a
non-specific (Ns) shRNA. Both DNA constructs contained shRNA and EGFP. Transfected cells were visualized and analyzed using transmitted
light (TL) and fluorescent microscopy (FM).
Result - Were the Cells Successfully Transfected with pEGFP-C3?

13. Results - Flow cytometry
Fig 6. Flow cytometry histogram of Non specific construct and specific construct.
A culture of P19 transfected cells, with vectors containing DNA sequence to transcribe ShRNA against FMNL2 RNA. Non-specific ShRNA was used
as a control. The X-axis represents the logarithm of EGFP fluorescence intensity from individual cells; 100
is the threshold to discriminate between
background fluorescence and transfected cells expressing EGFP. The percentage represents the total count which were considered successfully
transfected cells. 13
13.81% 12.60%

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Fig 7. RT-PCR for semi-quantitative analysis of FMNL2 expression.
RT-PCR was used to semi-quantitatively measure the expression of FMNL2 under specific and non-specific shRNA regulation in P19 cell lines.
The PCR products were analyzed using ethidium bromide stained gel electrophoresis. (A) Gel electrophoresis results of group W1. (B) Gel
electrophoresis results of group W7. RT-PCR was used to semi-quantitatively measure expression of FMNL2 in specific (Sp) and non-specific (Ns)
shRNA experiments. A negative control without a cDNA construct was also added, as well as a positive control containing a prepared cDNA.
FMNL2 expression was visualized between 600 and 700 bp, while GAPDH expression was measured as a loading control at roughly 300 bp.
Ratio of non-specific vs specific calculated using W7 at 25 cycles, standard deviation (std) generated using W2, W4, W7, W8 and W10 results.
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Results - Effects of shRNA on FMNL2 Expression
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Sample FMNL2/GAPDH
Non specific 1
Specific ± std 0.89 ± 0.15
A. B.

15. Discussion - Summary
Was it possible to interfere FMNL2 RNA with a ShRNA construct, using a PCR-based strategy ?
● High primer specificity
● Transfection efficiency = 12.6%
● Decrease in FMNL2 RNA = 11%
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16. Discussion - Related Research
Average transfection efficiency for P19 cells.
● 30 to 50 % transfection efficiency (Yu et al, 2005)
● Our transfection efficiency 12.6 %.
Decrease in expression of FMNL2
● Knockdown 87.13 % of FMNL2 in TIME cells(Mumal I., 2016)
● Our expression decrease is 11%.
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17. Discussion - Future Research
Rescue experiment
○ Supplement cells with exogenous FMNL2
○ Is there some other protein working with FMNL2
More in depth into altered morphology of cell contact.
○ Fluorescence microscopy
○ Polymer specific dye
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18. References
Breitsprecher, D., & Goode, B. L. (2013, January 1). Formins at a glance. Journal of Cell Science, Vol. 126, pp. 1–7. https://doi.org/10.1242/jcs.107250
FMNL2 formin like 2 [Homo sapiens (human)] - Gene - NCBI. (n.d.). Retrieved November 14, 2019, from https://www.ncbi.nlm.nih.gov/gene/114793
Grikscheit, K., Frank, T., Wang, Y., & Grosse, R. (2015). Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1. Journal of Cell Biology, 209(3), 367–376. https://doi.org/10.1083/jcb.201412015
Grobe, H., Wüstenhagen, A., Baarlink, C., Grosse, R., & Grikscheit, K. (2018). A Rac1-FMNL2 signaling module affects cellcell contact formation independent of Cdc42 and membrane protrusions. PLoS ONE, 13(3).
https://doi.org/10.1371/journal.pone.0194716
Kühn, S. ;, Erdmann, ;, Kage, F. ;, Block, J. ;, Schwenkmezger, L. ;, Steffen, A. ;, … Geyer, M. (2015). The structure of FMNL2-Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation. Item type Article.
Nature Communications. https://doi.org/10.1038/ncomms8088
Moore, C. B., Guthrie, E. H., Huang, M. T. H., & Taxman, D. J. (2010). Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods in Molecular Biology (Clifton, N.J.), 629, 141–158.
https://doi.org/10.1007/978-1-60761-657-3_10
Mumal I. (2016) The Role of Formins in Endothelial Adherens Junction Regulation( Master dissertation) University of Ottawa, Ottawa, Canada
dx.dio.org/10.20381/ruor-5310
Péladeau, C., Heibein, A., Maltez, M. T., Copeland, S. J., & Copeland, J. W. (2016). A specific FMNL2 isoform is up-regulated in invasive cells. BMC Cell Biology, 17(1). https://doi.org/10.1186/s12860-016-0110-z
shRNA Process and shRNA Diagram | Sigma-Aldrich. (n.d.). Retrieved November 16, 2019, from https://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/library-information/shrna-process-diagram.html
The different facets of actin cytoskeleton remodeling in migrating T... | Download Scientific Diagram. (n.d.). Retrieved November 18, 2019, from
https://www.researchgate.net/figure/The-different-facets-of-actin-cytoskeleton-remodeling-in-migrating-T-cells-Represented_fig3_284228226
Yu et al, (2005) Use of Short Hairpin RNA Expression Vectors to Study Mammalian Neural Development. Methods in Enzymology, Academic Press, 392, 86-199.
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19. Any Questions?
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