Dalle mappe al progetto genoma (Snustad e saggiolab). 2. Malattie genetiche,
clonaggio dei geni,terapia genica, transgenici (Snustad e saggiolab). 3. Giornata
...
Myc (cMyc) is a protooncogene, which is overexpressed in a wide range of human cancers. When it is specifically-mutated, or overexpressed, it increases cell proliferation and functions as an oncogene. Myc gene encodes for a transcription factor that regulates expression of 15% of all genes
1. Dalle mappe al progetto genoma (Snustad e saggiolab) 2. Malattie genetiche, clonaggio dei geni,terapia genica, transgenici (Snustad e saggiolab) 3. Giornata speciale sul Nobel alla Medicina 2007(saggiolab) 4. Junk DNA (saggiolab) 5. Eredita’ extra-cromosomica (Snustad e saggiolab) 6. Genetica dei tumori (Snustad e saggiolab) 7. Manipolazione dei genomi e clonazione (saggiolab) 8. Genetica di popolazioni (R. Petrucci, Snustad) 9. Regolazione espressione genica (Snustad) • Esercitazioni bonus (17/12, 19/12, 7/1, 9/1, 14/1). Riempire scheda/frequenza obbligatoria • Esonero scritto 23 gennaio 2008 • Verbalizzazione dopo l’esame del 6-2-08 (aperto anche ai non-frequentanti).
chi vive di vili smorzate muore di vili smorzate Peanuts
Manipolazione dei geni (in vitro e in vivo) vs manipolazione dei genomi
Animal Cloning - Dolly 1996
Sheep cloned by nuclear transfer from a cultured cell line. 1996 Nature Campbell et al UK •Cells from sheep embryonic disc -> culture -> epithelial line •Diploid nuclei transferred in enucleated metaphase II oocytes •Embryos implanted •morula frequency: 11-16% •Fetus frequency 100% •5 birth/8: 2 died at birth, 1 at 10 days, 2 ok. •Microsatellite analysis of cell line, fetus and lambs showed that all derived from a single cell population
Microsatellite analysis of cell line, fetus and lambs showed that all derived from a single cell population
Microsatellite FCB266
Random lambs
Cell line Cloned lambs
Identical patterns
Science december 1997
Human Factor IX Transgenic Sheep Produced by Transfer of Nuclei from Transfected Fetal Fibroblasts Angelika E. Schnieke, * Alexander J. Kind, William A. Ritchie, Karen Mycock, Angela R. Scott, Marjorie Ritchie, Ian Wilmut, Alan Colman, Keith H. S. Campbell UK
Classical approach for Transgenic mice
• DNA microinjection in the pronuclei of fertilized oocytes •5% of the animals integrate the transgene in the chromosome •Transgene expression variable •Germline transmission variable
Experimental procedure using animal cloning
• FactorIX: factor for blood coagulation; deficient in Hemophilia B: treated usually with human plasma (contamination problems) •DNA:Factor IX under control of ovine beta-lactoglobulin promoter •Lipofectamine-transfected in primary strains of ovine cells (fetal fibroblasts) •Selected stable transfectants (neo) •Cells brought to G0 (by serum deprivation) (epigenetics?) •Transfer of nuclei in enucleated oocytes
Efficiencies analogous to those of non transduced cells
Table 1. PDFF5 (non-tr) Reconstructed embryos 82 morulae or blastocysts 5 (6.1%) Embryos transferred 5 Recipients 2 Pregnancies at day 60 2 Fetuses at day 60 (% of embryos) 3 (60%) born (% of embryos) 1 (20%) (% live lambs vs embryos) 1.22%
PDFF2 pool
PDFF2-12
PDFF2-13
224 22 (9.8%) 22 9 4 4 (18.2%) 3 (13.6%) 1.34%
89 112 19 (21.4%) 23 (20.5%) 19 21 7 6 4 1 6 (31.6%) 1 (4.8%) 2 (10.5%) 1 (4.8%) 2.25% 0.89%
Presence of FIX DNA. Not in all lambs.
Blood from lambs and southern MIX: transfected FIX BLG: internal gene NEO: transf selection marker
NB: funziona meglio se uso nuclei derivati dai cloni
Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing (rabbits and cattle) Ludger Grosse-Hovest * , Sigrid Müller , Rosa Minoia , Eckhard Wolf ¶, Valeri Zakhartchenko ¶, Hendrik Wenigerkind ¶, Caroline Lassnig || **, Urban Besenfelder **, Mathias Müller || **, Simon D. Lytton , Gundram Jung * and Gottfried Brem || PNAS 2003-USA (Texas)
mg/ml production of Abs
Protocol •VH-VL genes specific for melanoma antigen •Introduced in fertilized rabbit oocytes or bovine fetal fibroblasts •Transgenic animals (by nuclear transfer in the case of bovine fibroblasts) •Test blood activity CD28 on T cells
Ab
Melanoma antigen
T cell proliferation and tumor cell killing
Fig. 2. Purification of bi-scFV r28M and determination of binding capacity: calf blood
Protein expression
Protein cell binding activity
Grosse-Hovest, Ludger et al. (2004) Proc. Natl. Acad. Sci. USA 101, 6858-6863
Copyright ©2004 by the National Academy of Sciences
Fig. 3. Proliferation of human PBMC and killing of tumor target cells incubated with bi-scFV r28M
Prolif.
Killing
Killing
Grosse-Hovest, Ludger et al. (2004) Proc. Natl. Acad. Sci. USA 101, 6858-6863
Copyright ©2004 by the National Academy of Sciences
Nature 407, 86 - 90 (07 September 2000); doi:10.1038/35024082
Cloned pigs produced by nuclear transfer from adult somatic cells IRINA A. POLEJAEVA*, SHU-HUNG CHEN*, TODD D. VAUGHT*, RAYMOND L. PAGE*, JUNE MULLINS*, SUYAPA BALL*, YIFAN DAI*, JEREMY BOONE*, SHAWN WALKER*, DAVID L. AYARES*, ALAN COLMAN† & KEITH H. S. CAMPBELL†‡ * PPL Therapeutics Incorporated, 1700 Kraft Drive, Blacksburg, Virginia 24060, USA † PPL Therapeutics, Roslin , Midlothian EH25 9PP, UK ‡ Present address: University of Nottingham, School of Biosciences, Sutton Bonington, Leicestershire LE12 5RN, UK
More complex procedure to clone pigs
Why doing it? You can control DNA content
Microsatellite analysis: table indicates PCR fragment size
granulosa
piglets
Surrogate mother
zygote
NB Low polymorhpism because of inbred animals
Nature 21 February 2002
Cell biology: A cat cloned by nuclear transplantation Shin et al.
USA Felis domesticus
Experimental procedure and results 1st attempt: Fibroblast cells from oral mucosa expanded in culture Fused to enucleated metaphase II oocytes 188 fusions -> 82 embryos -> one pregnant cat -> no birth 2nd attempt: Cumulus cells (granulosa cells) from adult cat expanded in cultures 2 cloned embryos from cumulus cells implanted 3 cloned embryos from fibroblast cells implanted 1 birth
In the same cat
One kitten out of 82+3+2 implanted embryos
donor
Surrogate mother and cloned kitten (color is not fully genotype determined)
Analysis of seven unlinked highly polymorphic felinespecific microsatellite loci confirms that the kitten is a clone
Italia 2003: clonaggio di un cavallo da cellule della pelle
The cells used for cloning were derived from a skin biopsy of the mare. The enucleated oocytes were obtained by in vitro maturation and removal of the nucleus from oocytes recovered from horse ovaries from an abattoir. After embryo reconstruction by micromanipulation and cell fusion, the cleaved embryos were cultured in vitro to the blastocyst stage. Two of the derived embryos were transferred into the mare donor of the cell line. A single pregnancy was established and was carried to term with the birth of a female foal who is genetically identical to her surrogate mother. Galli et al Nature 2003
…Il genetista Cesare Galli che per primo al mondo ha clonato un toro partendo da una cellula del sangue, si dice pronto a sperimentare la clonazione di Varenne -il cavallo che ha riportato il trotto italiano ai vertici internazionali- se cio' gli fosse chiesto. "Puo' essere un obiettivo e certamente ci piacerebbe -conferma lo scienziato che dirige il Laboratorio di tecnologie della riproduzione presso il Consorzio per l'incremento zootecnico di Porcellasco, alla periferia di Cremona. Non abbiamo pero' ancora ricevuto una richiesta in tal senso….
Nature 04 March 2004
Mice cloned from olfactory sensory neurons KEVIN EGGAN1,*,†, KRISTIN BALDWIN2,*, MICHAEL TACKETT1, JOSEPH OSBORNE2,†, JOSEPH GOGOS2, ANDREW CHESS1, RICHARD AXEL2 & RUDOLF JAENISCH1 1 Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts
Institute of Technology, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA 2 Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, New York 10032, USA
Post-mitotic (termially differentiated) cells can be reprogrammed to totipotency? •Regulatory proteins •Covalent modifications in chromatin proteins or Dna (epigenetic..) •DNA rearrangements (neurons: contributes to neuronal diversity?) e.g. each of the 2,000,000 cells in the olfactory epithelium expresses only one of 1,500 odorant receptor genes
Con sistema di Cre-loxP GFP marcano i neuroni olfattori: incrocio fra topi olfactory marker protein (OMP)-cre e topi loxP stop gfp.
L’ espressione di cre elimina sito di stop prima di GFP. Cosi’ omp che controlla cre a sua volta controlla indirettamente l’espressione della gfp
Green: gfp; red: cre or progenitors or BrdU or dividing cell marker; blue: nuclear marker Post mitotic: no BrDu, yes GFP (e)
Nucleus from olfactory neurons in enucleated oocytes
Olfactory epithelium Wt expression of olfactory neurons indicates reversibility of DNA rearrangements
Raelian leader says cloning first step to immortality February 12, 2004
Brigitte Boisselier, scientific director of Clonaid, said the first cloned human baby was born Thursday
Originally published in Science Express on 12 February 2004 Science, Vol 303,
Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst Woo Suk Hwang,1,2* Young June Ryu,1 Jong Hyuk Park,3 Eul Soon Park,1 Eu Gene Lee,1 Ja Min Koo,4 Hyun Yong Jeon,1 Byeong Chun Lee,1 Sung Keun Kang,1 Sun Jong Kim,3 Curie Ahn,5 Jung Hye Hwang,6 Ky Young Park,7 Jose B. Cibelli,8 Shin Yong Moon5*
KOREA-USA Therapeutic cloning:nucleus of somatic cell in oocyte, ES cell purified from preimplantation embryo
Experimental procedure
Autologous Somatic Cell Nuclear Transplantation Cumulus cells and enucleated oocytes MII, Fusion Artificial stimulus for development
SCNT-ES forming colonies
A and B enucleation C to E colony of SCNTES cells F nestin IF (primitive neuroecotderm differentiation marker) G caryotyping
SCNT-ES express ES markers
SCNT-ES in immune-deficient mice generate teratomas that include different tissues. A neuroepith. B retinal epithelium C bony differentiation D cartilage E smooth muscle and connect
DNA fingerprinting
Nature Medicine 26 May 2005 Profile: Woo-Suk Hwang Apoorva Mandavilli San Die go
In the West, Woo-Suk Hwang is an enigma. In his home country, he is a scientist with perhaps too much power. In either place, he is a stem cell star. 'hanul eul gamdong shikyeara' let's move the sky
Nature Medicine 12, 4 (2006) South Korean scandal rocks stem cell community
L’ultima barriera: la partenogenesi. ovvero Si deve partire da cellula somatica composta da un patrimonio genetico misto? Ricordiamoci dell’ imprinting
Nature 428, 860 - 864 (22 April 2004); doi:10.1038/nature02402
Birth of parthenogenetic mice that can develop to adulthood TOMOHIRO KONO1,3, YAYOI OBATA1,3, QUIONG WU1,3, KATSUTOSHI NIWA1,3, YUKIKO ONO1, YUJI YAMAMOTO2,3, EUN SUNG PARK4, JEONG-SUN SEO4,5 & HIDEHIKO OGAWA1,3 1 Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-
8502, Japan 2 Department of Applied Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan 3 Bio-oriented Technology Research Advancement Institution (BRAIN), Minato-ku, Tokyo 105-0001, Japan 4 MacroGen Inc, Chongno-Ku, Seoul 110-061, Korea 5 Department of Biochemistry, Seoul National University College of Medicine, ChongnoKu,
2 aploid sets of a maternal genome in a reconstructed oocyte but with differences in genes implicated in imprinting
SCNT (somatic cell nuclear transplantation) useful for • • • •
Cloning Stem cell production Epigenetic studies Drug production
Some concepts to think about
• No fertilization needed • Reversibility in the control of DNA expression • Imprinting?? • Future: use of specific genes to produce ES cells from differentiated cells, instead of the whole genome
