A conference presentation summarizing my research project on fibroblast differentiation to retinal cell types.

1. CONVERSION OF
FIBROBLASTS TO RETINAL
CELLS BY TRANSCRIPTION
FACTORS
Lucas Man
Xiang Lab

2. Creating retinal cells
• We hope that by activating combinations of retinal-lineage-specific
transcription factors in mouse embryonic fibroblasts (MEFs), we can
induce the MEFs to differentiate into retinal cell types
Scienceblogs Thermo Fisher Scientific, Inc.

3. Overview
Lentivirus-mediated transduction
3. Transfect packaging cells with
the viral vector and packaging
plasmids
4. Harvest virus particles
5. Create viral cocktails and infect
target cells
1. Clone factor by inserting
factor of interest into cloning
vectors
2. Sub-clone factor from 6. Immunostain to identify
cloning vector into virus presence of retinal-cell-
vector specific markers

4. The Factors
• 17 factors specific to retinal cell lineages were selected:
Completed lentivirus vectors: Still in progress:
Pax6 Rax/Rx Sox2 Six3/6 Meis1/2
Otx2 Foxn4 Crx Prdm1/Blimp1
Math5 Brn3b Isl1 Neurod1
Math3 Ptf1a NrI Tlx/Nr2e1

5. 1. Cloning Factor
• Ligate gene of interest into
cloning vector
• Transform E. coli with
cloning vector and culture
SystemBio

6. 1. Cloning Factor (Cont.)
• Purify plasmid from E. coli
colonies
• Cut plasmid at restriction
enzyme sites
• Send gene for sequencing

7. 1. Cloning Factor (Cont.)
• Use BLAST to compare sequencing results to known
sequence of gene of interest
Brn3b sequencing
chromatogram and BLAST
results

8. 2. Creating a Lentivirus
• Ligate gene into viral vector
• Transfect packaging cells
(293T cells) with viral
vector and packaging
plasmids (vsv-g and
pCMV)
• Culture packaging cells
and harvest supernatant
Kenyon College
Dept. of Biology

9. 3. Testing Virus
• Culture MEFs in control and experimental groups
• Control group is cultured in clean medium while
experimental groups are exposed to medium with virus
particles
• 2 experimental groups: Dox – and Dox +
Control Infection + Infection +
(no virus Dox – Dox +
or Dox in (virus in (virus in
medium) medium, n medium,
o Dox) Dox)

10. 3. Testing Virus (Cont.)
• Doxycycline (Dox)
• an antibiotic, a derivative of tetracycline
• Tet-On inducible expression system:
• Tetracycline transactivator protein (tTA) binds DNA at an operator and
activates a promoter, initiating transcription of nearby genes
• rtTA is a version of the tTA protein that only binds to the operator when
bound by Dox
• Introduction of Dox initiates transcription
• All our factors are coupled with a Tet-On switch, requiring
Dox for transcription activation

11. 3. Testing Virus (Cont.)
• After culturing MEFs for 4-5
days, lyse cells and purify
RNA
• Reverse transcribe mRNA to
cDNA
• PCR using gene-specific
primers to determine levels of
expression for gene of
interest
• Expected results:
Tubulin
• Control group: no to low level of
expression for the factor
• Dox – group: similar level of
expression as control group
• Dox + group: high level of
expression for the factor
• Tubulin as internal control

12. Summary
• Clone transcription factors
• Prepare factors into virus vectors for delivery into target MEFs
• Test viruses for infection competency
Going Forward
• Finish producing and testing all viruses
• Create virus cocktails with different combinations of factors
• Infect MEFs with virus cocktails
• Hopefully induce MEFs to differentiate into retinal cell types

13. Acknowledgements
• My mentor Min Zou
• The other graduate students and post-docs in the Xiang Lab
• Dr. Xiang
Questions?