2. What is the “big picture” question?
Can they derive fully reprogrammed equine induced pluripotent stem cells?

3. Induced Pluripotent Stem Cells
Patient
iPSCs
POU5F1, SOX2, NANOG, LIN28, KLF4, C-MYC
Patient Specific
Differentiated Cells

4. Why would you want to generate equine iPSCs?
Autologous Cell Therapy

5. Why would you want to generate equine iPSCs?
Drug Development
Species Specific
Cell Type Specific
Testing Toxicity
Studying Infectious Diseases
Abnormal Development

6. Figure 1.

7. Figure 1.

8. hESCs Karyotype in TeSR1?

9. Immunocytochemistry

10. Figure 2.

11. Figure 2.

12. What is RT-PCR?
Reverse Transcription Polymerase Chain Reaction is a process where RNA is reverse transcribed into complementary or cDNA and then undergoes PCR amplification typically for a target gene of interest.

13. Question 1. Why would you do RT-PCR?
Answer 1. To detect the presence of a specific RNA

14. Question 2. Why would you want to detect the presence of a specific RNA?
Answer 2. Lots of reasons!
Check the developmental state of a cell, tissue and/or animal
Check the expression of a transgene
Detect if animal is a carrier of a gene of interest

15. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Is not to be confused with Real Time Polymerase Chain Reaction (RT-PCR)

16. Advantages of RT-PCR Fast Simple Economical
Can be done in 1 day once sample is collected
Simple
Reagents come ready to use and optimized
Economical
$7/primer set

17. Major Steps in RT-PCR Isolate RNA Reverse transcribe RNA into cDNA
Amplify target sequence using PCR (Show Video)
Visualize and Image utilizing gel-documentation station

18. RT-PCR

19. Figure 3.

20. Figure 3.

21. Figure 3.

22. Figure 4.

23. Figure 4.

24. Figure 5.

25. Figure 5.

26. Figure 5.

27. What is the big picture conclusion?
Yes they derived fully reprogrammed equine induced pluripotent stem cells.

28. What were some of the flaws of the paper?
What do you think are some of the next steps?
Do you think that eiPSCs are a viable option for therapy?

29. RT-PCR Example Experiment: Question
Can hESCs differentiate (turn into) into sperm over 20 days in sperm media?

30. RT-PCR Example Experiment: Experimental Design
hESC
Day 0
Day 10
Samples
Day 20
Samples
Questions to ask yourself?
What types of controls do you need?
What genes do you want to look at?
How many replicates?

31. RT-PCR Results

32. Primer Design Pick a gene
Go to NCBI Website (
Select Nucleotide option from drop down panel
Type in gene of interest and select search
Click on correct gene variant of interest and species
Select Pick Primers from right panel
Select PCR product length (typically bp)
Select melting temperature (between 53 °C and 63 °C ; >65 °C get excessive secondary structure formation)
Select exon junction span: exon-exon junction

33. Hints for Ordering Primers
Always order more than 1 primer set
Can order through the Georgia Genomics Facility on Campus (

34. Let’s Design Some Primers