Lecture 20 Thursday November 22 2007

Reproductive cloning

  • dolly
  • cloning embryos
  • worked well in cattle and has been done for years
  • nucleous extraction
  • replacement with chosen DNA

therapeutic cloning

  • making stem cell lines
  • this enables the regeneration of organs

fetal stem cells

  • not as good as embryonic stem cells
  • won't last forever

adult stem cells

  • we don't have very many so finding them is difficult

existing human embryonic stem cell lines

  • nov ery well characterized
  • what we need is patient specific stem cell lines

patient specific stem cell lines

  • these are good because they

ethical source o huan embryonic stem cells

  • distribution of embryos
  • source of eggs

derivation

  • sperm + egg = embryo
  • eight cell stage
  • blastocist

standard nuclear transfer

altered nuclear transfer (how you get around not producing an embryo)

  • somantic nuclei genetically modified to impair embryo 's ability to develop
  • cdx2 is a transcription factor
  • with out cdx2 you can no implant a stem cells
  • can still isolate embryonic stem cell
  • there are clear problems with this theory

parthenogenesis

  • development of embryo from an egg without fetilization by sperm
  • getting an embyo from an egg without fertilization
  • this embryo will not develop - you need male genes

meiosis

  • Two copies of a single chromosome
  • line up to form bivalents
  • meiosis one
  • each cell has one chromosome that is duplicated
  • first polar body is thrown away
  • this is because of
  • the egg is arrested after meiosis one
  • stop it from going through second miotic division with drugs
  • diploid cell will not develop but will give off stem cells

genomic imprinting

  • in a few cases, gene expression depends which parent the allele came from
  • paternal and maternal alleles are expressed differently
  • controlled by modifications to the DNA that do not affect

imprinting process

  • erase genes
  • the result is that the males contribution in the parthogenesis - this alters gene expression in the embryo is deformed

organismically dead embryos

  • needs OCT4 to divided beyond a certain stage
  • however, at the point just before death stem cells are extracted

chromosomally abnormal embryos

  • self normalization - genes fix themselves to create normal embryonic stem cells

single blastomere biopsy

  • used today in medicine
  • preimplantation genetic diagnnosis
  • has the potential to damage the embryo
  • remove a single cell from the embryo
  • test it for disease
  • wait for it divide by culturing it in vitro
  • you can them make a stem cell line

somantic cell differentiation

  • change a differentiated somantic cell into a pluripotent stem cell

dedifferentiation

  • can you force a stem cell to de differentiate
  • you stick them in a dish and reprogram it
  • it loses its differentiated properties

epigenetics

  • changing DNA without changing DNA sequence
  • involves chromatin remodeling
  • so you want to take permanently turned off genes and turn them back on
  • you have to remove epigenetic marks
  • it is the best way to go but is difficult

Human ESC lines

  • 2004 published the creation of the first cloned human embryos
  • he reported that he made 10
  • no cloned human embryos

last week

  • dr. shouklhat mitalipov at oregon health & science university
  • cloned macque embryos
  • using SCNT (skin cells)
  • extracted stem cells from some of the cloned monkey embryos
  • able to develop into mature heart and nerve cells in the laboratory
  • took 10 years and 15000 eggs

Doll scientist abandons cloning - November 2007

  • prof shinya Yamanaka of Kyotto Univeristy, Japan
  • no eggs, or embryos
  • generically modified human adult cells to make them almost as pluripotent as stem cells
  • adding back genes allowed the genes to resume expression
  • made 10 pluripotent lines from culture of 50, 000 skin
  • used 4 transcription factor genes (MYC - is an oncagene that could turn into cancer) to add back
  • adding extra copies of the gene
  • Scientists are allowed to do this - no embryo's

adding back doesn't always work they have not refined

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