BOIC 095 Altering Long Non-coding RNA Expression & Assocaited Impacts On Osteoblast Differentiation

Question 1

Mimi Falcone with Dr. Coralee Tye in the Stein/Lian Lab

Answer 1

1

Question 2

Noncoding RNA

Answer 2

• 75% of genome is transcribed by mRNA; only 2% encodes protein
• Remainder consists of noncoding RNAs (ncRNA) involved in various regulatory roles
• Focus on long noncoding RNAs (lncRNA): all ncRNA transcripts longer than 200 nucleotides
  > Many diff function in protein formation (6)
• e.g. enhancer RNA, transcriptional activation, transcriptional repression, scaffolding protein for chromatin remodeling complexes

Question 3

Lit

Answer 3

• 2014: human genome project identified 24,489 lncRNA transcripts involved in regulating gen expression
• Very few individual lncRNA involved in osteoblasts differentiation

Question 4

Ca dkdate lncRNAs of interest

Answer 4

• Dancer

- Mir22hg

Question 5

Hypothesis

Answer 5

• Multiple lncRNAs regulate osteogenesis thru various mechanisms
• Aim: investigate the complexity of lncRNA function in differentiation of mesenchymal stomal cells

Question 6

Methodology

Answer 6

• Alter lncRNA expression by CRISPRa & CRISPRi
• Infect bacterial plasmids into virus
• Infection of altered plasmids into hMSCs
  (Mesenchymal stomal cells > (differentiated) > pluripotent stem cells
• Differentiation of hMSCs
  (21 day: 0 undifferentiated, 7 committed, 14 ECM production, 21 mineralization)
• Employ functional tests
  (Alkaline phosphatase = enzyme histochemical stain for osteoblasts)

Question 7

Data / Results

Answer 7

• W/O success

- in process of altering methods to grow up a stable cell line

Question 8

Future

Answer 8

• Use puromycin selection & flow cytomegalovirus to produce confluent cell populations contains the altered plasmids
• Use PCR to validate primers to make sure plasmid alteration via CRISPR is effective
• Use qPCR to look at expression levels of genes associated with specific lncRNA

Question 9

Applications

Answer 9

• lncRNAs are potential targets for drugs & other medical therapies for bone-related diseases
• E.g. use lncRNA to reinstate functional osteoblasts imprinting in patients

Question 10

How to get involved in research

Answer 10

• Identify ur own interests
• Search for a UVM professor / faculty member conducting similar research
• Reach out: ask more about their research, inquire abt shadowing
• Talk to your peer: upper class men are a great resource