1. Eukaryotic cell organelles http://www.jeffdonofrio.net/DNA/DNA_Analysis.html

2. Inheritance Each cell has MANY mitochondria Oocytes have many mitochondria Sperm mitochondria are specialized, jettisoned at fertilization Mitochondria are only MATERNALLY inherited

3. Mitochondrial Function Energy production- via Krebs Cycle  ATP Glucose  pyruvate = 2 ATP Pyruvate  CO 2 and H 2 0 = 34-36 ATP Calcium homeostasis Role in apoptosis Role in blood and hormone synthesis Main tissues with high energy needs Brain, heart, liver, muscles, respiratory, endocrine

4. Human mitochondrial genome Nageswara R. Madamanchi, and Marschall S. Runge Circulation Research. 2007;100:460-473 Copyright © American Heart Association, Inc. All rights reserved. 37 genes ~17,000 bp 13 polypeptides, 22 tRNAs, 2 rRNAs).

5. Mitochondrial Dysfunction Loss of motor control Muscle weakness Seizures Visual and auditory problems Poor growth Cardiac disease Liver disease Developmental delays Susceptibility to Infection

6. Variants in mitochondrial genes variety of diseases 1 in 200-250 live births has a potentially pathogenic mitochondrial DNA mutation Often delayed onset Not directly lethal 45 listed on site- United Mitochondrial Disease Foundation diseases of mitochondrial function -www.umdf.orgwww.umdf.org

7. Could Replacement of Mom’s Mitochondria Repair the Problem? During Female embryonic development After puberty At each cycle

8. Overview of spindle- chromosomal complex transfer Video mms://media.ohsu.edu/host/hosp/replacement.wmv Healthy Baby

9. IVF required for embryo development (Photo Credit: Mr. J. Conaghan) http://stemcells.nih.gov/info PlacentaEmbryo

10. Mitochondria appear everywhere except near the chromosomes

11. Visualization of the spindle during enucleation avoids damage

12. Spindle-chromosomal complex successfully isolated

13. Karyoplast contains ~1.5% volume compared to a cytoplast

14. Karyoplast inserted into perivitelline space Karyoblast with patient’s spindle

15. Fusion using Sendai virus extract yields metaphase II chromosomes

16. Movie mms://media.ohsu.edu/host/hosp/replacement.wmv

17. Chronology of innovation Late 1990s: Cytoplasmic transfer leads to ~100 births worldwide 2001: FDA halts cytoplasmic transfer September 2009: Mitochondrial gene replacement in primate offspring and embryonic stem cells May 2010: Pronuclear transfer in non-viable human embryos October 2012: Mitochondrial gene replacement in human embryos to blastocyst stage January 2013: Two additional studies demonstrating feasibility in human

18. Success rate: 4 healthy births Mito and Tracker

19. 15 spindle-transfer embryos implanted into 9 recipients

20. Sequencing confirms mixed parentage

21. Pronuclea r transfer is an alternate approach

22. Some mitochondria may also be transferred