C336 Molecular Biology in Science and Medicine 2006

WD Richardson: Genetic manipulation in mammals - 4 lectures

• Transgenic animals. What are they? Why are they important?

                    - research tools for studying gene function and regulation in an in vivo context

- living "factories" for production of proteins that are important in biotechnology or biomedicine ("pharming")

· Mammalian cloning as a way of expanding a transgenic animal colony rapidly

• Transgenic mice by pronuclear injection. An example of their use - studying the transcriptional regulation of the platelet-derived growth factor receptor gene (locations of essential cell-type specific enhancer elements)

• Derivation of embryonic stem (ES) cells from the iner cell mass of the mouse embryo. Totipotent nature of ES cells re-injected into host blastocyst. Knocking out gene function in ES cells: targeting vectors, homologous recombination, positive/negative selection with neo/G418 and HSV-TK/gancyclovir. Generating whole mice from ES cells.

• Tissue- or cell-specific knockouts using the Cre-lox recombination system of bacteriophage P1; "flox" the gene of interest via the ES cell route, express Cre under a tissue-specific gene promoter in transgenic mice (knock-in or BAC vector), cross the two mouse strains.

• Temporal control of gene knockout: Cre-estrogen receptor (Cre-ER) fusion that is activated only in presence of hormone (a mutated form of ER is used that binds tamoxifen, a synthetic version of estrogen, but not estrogen itself). This allows tissue-specific, inducible gene knockout with a single transgene.

• Other applications of the Cre-lox genetic switch: fate mapping the embryo with a Cre-activated reporter (lacZ or GFP), cell ablation with diptheria toxin A chain

Reading:

mouse embryology, ES cells, chimeric mice, knockouts:  Scott F Gilbert, Developmental Biology, 6th Edition (Sinauer Associates Inc., Sunderland, Massachusetts) 2000. pp97-100.

example of gene targeting using ES cells: Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, Yagishita N, Matsui D, Koga Y, Itoh N, Kato S. (1999). Fgf10 is essential for limb and lung formation. Nature Genetics 21, 138-141

conditional knockouts:  1) Lobe C.G. and Nagy A.(1998). Conditional genome alteration in mice. Bioessays 20, 200-208. (review).     2) Malleret G, Haditsch U, Genoux D, Jones MW, Bliss TV, Vanhoose AM, Weitlauf C, Kandel ER, Winder DG, Mansuy IM. (2001). Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin. Cell 104, 675-686.

fate- mapping with Cre-lox:   Fogarty M, Richardson WD, Kessaris N. (2005). A subset of oligodendrocytes generated from radial glia in the dorsal spinal cord. Development 132, 1951-1959. [PDF]

cell ablation with diphtheria toxin(DTA) transgene: Kessaris, N., Fogarty, M., Iannarelli, P., Grist, M., Wegner, M. and Richardson, W.D. (2006). Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage. Nat. Neurosci. 9, 173-179. [PDF]