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

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

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

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

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Figure 1.

hESCs Karyotype in TeSR1?

Immunocytochemistry

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Figure 2.

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.

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

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

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

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

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

RT-PCR

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Figure 3.

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Figure 5.

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

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?

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

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?

RT-PCR Results

Primer Design Pick a gene Go to NCBI Website (http://www.ncbi.nlm.nih.gov/pubmed/) 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 200-800 bp) Select melting temperature (between 53 °C and 63 °C ; >65 °C get excessive secondary structure formation) Select exon junction span: exon-exon junction

Hints for Ordering Primers Always order more than 1 primer set Can order through the Georgia Genomics Facility on Campus (http://dna.uga.edu/)

Let’s Design Some Primers