Piezo1 Gene Knockdown in Primary Mouse Cells

Yan Pacheco

Authors:  Yan C. Pacheco, Jacob Griffith, and Kyle Allen

Faculty Mentor:  Kyle Allen 

College:  Herbert Wertheim College of Engineering

Abstract

Osteoarthritis (OA) is a progressive disease characterized by joint degeneration, resulting in varying degrees of pain. However, the role of mechanically activated ion channels in OA etiology is not fully understood. The Piezo1 gene encodes for a protein that links mechanical forces to biological signals. The focus of this study was to quantify the effect of a Piezo1 knockdown on relative expression levels of extracted fibroblasts and chondrocytes. Menisci and the femoral head were extracted from each leg of four mouse cadavers. For fibroblast extraction, menisci were placed in a solution of Dulbecco’s Modified Eagle’s medium (DMEM), 1% penicillin/streptomycin, 0.2% collagenase II, and 0.2% collagenase I. Chondrocytes were extracted by digesting the femoral head in a solution containing DMEM, 1% penicillin/streptomycin, and 0.2% collagenase II. The tissues were strained (40 µm cell), centrifuged, and plated in two separate plates. In one plate, Piezo1 was knocked down, using a lentivirus vector. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed using three different piezo1 primers and GAPDH. Knockdown efficiency was determined by comparing relative expression levels between the two plates. This study establishes the framework for our future studies investigating the effect of piezo1 knockdown on OA progression and severity.

Poster Pitch

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Poster

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Talline
Talline (@guest_218)
1 year ago

nice job, Yan!

Yan
Yan (@guest_6474)
Reply to  Talline
1 year ago

Thank you Dr. Martins!

Valerie Lantigua
Valerie Lantigua (@guest_1452)
1 year ago

Great work as always Yan! Your poster looks so clean and polished, I love it

Dennis Le
Dennis Le (@guest_1796)
1 year ago

What was the diluting solution described in the effiiciency curve? How does that reflect on the efficiency result graphs? What kits do you used to isolate the RNA?

Yan
Yan (@guest_3696)
Reply to  Dennis Le
1 year ago

The dilutions were done in DNase free water, provided by the cDNA kit we also used. It was the same that was used for all the PCR reactions in order to standardize our results. As far as RNA isolation kits we used a Quiagen RNeasy Mini Kit.

Nicole Sarna
Nicole Sarna (@guest_2740)
1 year ago

Great job, Yan! Thanks for sharing 🙂

Joshuah Demetrius
Joshuah Demetrius (@guest_3252)
1 year ago

Loving the lights in the background! Extra spice for the pitch!

Julia Bittencourt
Julia Bittencourt (@guest_4990)
1 year ago

The bowtie ??

Great job, Yan!

David Julian
David Julian (@guest_5230)
1 year ago

Good job, Yan. You’ve certainly raised the bar sartorially! What else other than pipetting errors could have caused an apparent primer efficiency greater than 100%?

Zachary Player
Zachary Player (@guest_5700)
Reply to  David Julian
1 year ago

I’m also curious about the >100% efficiency. Other than that, looks great!

Yan
Yan (@guest_5922)
Reply to  David Julian
1 year ago

Thank you Dr. Julian. Other possible reasons could be that the cDNA used could have been slightly contaminated or that ethanol/other solvents could have gotten inside the wells before the were loaded onto the PCR machine. There could also have been some reverse transcriptase inhibition

Yasmin
Yasmin (@guest_6056)
1 year ago

Incredible job Yan!!! I love the way you organized your poster. So why do you think the primer in the green graph did not bind it well?

Yan
Yan (@guest_6276)
Reply to  Yasmin
1 year ago

Thank you Yasmin! I think there could have been a couple things that caused the poor melt curves. First, the primer had a slightly different melting temperature than the rest. Secondly, I think the sequence was most likely not specific enough for the gene. This is why we wanted to try 3 different primers instead of just choosing one at random.