Post on 12-Mar-2020
Development of stem cell technologyfor livestock species in Taiwan
Lih‐Ren Chen, PhDLivestock research Institute, Council of Agriculture
Taiwan
(Winslow, 2001)
Chen, L. R., J. P. Wang and M. C. Wu, 1991. In vitro culture of mammalian embryonic stem cells : II. In vitro culture of blastocyst- derived cells in mouse and pigs. J. Chin. Soc. Anim. Sci. 20(3):326-339.
Shim, H., A. Gutierrez-Adan, L. R. Chen, R. H. BonDurant, E. Behboodi and G. B. Anderson. 1997. Isolation of pluripotent stem cells from cultured porcine primordial germ cells.BOR 57:1089-1095.
Chen, L. R., Y. L. Shiue, L. Bertolini, J. F. Merdrano, R. H. BonDurant and G. B. Anderson, 1999. Establishment of plurpotent cell lines from porcine preimplantation embryos. Theriogenology 52:195-212.
Embryo Splitting
Micromanipulator
“The cloning of mammals…………..…. is biologically impossible.”
James McGrath and Davor Solter, Science 1984.
• Ian Wilmut• Created the
first true clone, the Dorset ewe Dolly
Dolly, the sheep
1996 Dolly the sheep
1998 Mouse, Cow
1999 Goat
2000 Pig
2002 Cat, Rabbit, African wild cat
2003 Mule, House, Rat, Banteng,
Deer, Rat
2005 Dog
Somatic clone animals
C D
A B
0901/2001, 畜寶 1016/2001 0122/2002
寶吉與寶祥, 0705/2003
如意, 0227/2003
0227/2003, 1211/2003, 0116/2003, 0122/2004
0217/2005
V.S• Growth
• Reproduction
• Production
• Gene expression
• Easy to manipulate• Low efficiency
• Variety gene expression level
• nearly impossible for gene targeting
• Complicated manipulation
• Low efficiency
• Uniform gene expression level
• Possible for gene targeting
Vectors for genetic modification
Preparation of nuclear donor cells
Ear fibroblasts
Frozen
Gene Transfer
Subcloning
寶鈺, 0310/2004
0425/2005
Transfection
Incubation and Passage
Selection
Somatic cells donor
Cloning
Nuclear transfer
Limited passage number
Possible damage in X’some
RA
(Yang et al., 2006)
Nestin +
NF68 +
MAP2+
ChAT+
TH+
(Yang et al., 2006)
++++AP activity
++++Oct‐4+++‐TRA‐1‐81+++‐TRA‐1‐60+++‐SSEA‐3/4‐‐‐+SSEA‐1
pEShESrESmESItem
+??+Chimera formation
++++EB formation
Flat, tightaggregates
Flat, loose aggregates
Flat, loose aggregates
Tight, rounded, multi‐layer clumps
Growth characteristics
‐+/‐‐+LIF for selfrenew
++++/‐Feeder dependency
pEShESrESmESItems
Cloning ES Tech
Solter and Gearhart, 1999
Differentiation induction
Isolation and culture of ES cells
Pig Somatic cell
Therapeutic cloning in pig model
Immunologicallycompatible transplant for cell therapy
IVM pig oocyte Enucleation
Transfer of donor cell Electrofusion
LPE-206-1
Donor cell
8(17.3)46(74.1)62(51.6)120
BlastocystCleavedSuccessfully fused
Used for SCNT
Number and(%)of fused embryos
Number and(%)of oocytes
Pig somatic cells
Dominko et al., 1999. Biol. Reprod. 60:1496-1502.
Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic nuclei from various mammalian species.
Hybrid Embryo development
Hatching blastocyst at day 7 Hatched blastocyst at day 9
Morula at day 5 Early Blastocyst at day 6
11 (50%)
2‐16 cells
Cleavage (%)(72%)
Adult female buffalo fibroblast
Donor cell
2 (9%)3 (13.6%)
BlastocystMorula22 (84%)2627
fused (%)Nuclear transferoocytes
Stem cells
Differentiated adult cells
Human/mouse
somatic cells
streptolysin-O Remove
Stem
cell
extrac
tion
Oocyte/
Stem cell extract
Resealing plasma membrane
Morphology change of reprogrammed mouse and human fibroblasts
b
β-actinOct-4
Nanog
mES Reprogrammed hF
β-actin
Oct-4
NanogPECAM-1
PECAM-1
Reprogrammed mF
β-actin
Oct-4Nanog
PECAM-1
hF
AP+
SSEA-1+
Acknowledgement• Livestock Research Institute
– Dr. JJ Tailiu– JR Yang– TY Kuo– FH Chu– YH Chen– Dr. JW Shiau
• Buddhist Tzu Chi Medical Center– Dr. SZ Lin– CH Liao
• National Chung HsinUniversity– Dr. CM Chen– Dr. MC Huang
• National Sun Yat‐sen University– Dr. YL Shiue
• National Chung Kung University– Dr. C Tai– CH Kang– FT Lu
• National Taiwan University– Dr. WTK Cheng
• National Pintong Scientific U.– Dr. PC Sheen
• University of Connecticut– Dr. X Yang – Dr. X Tian, – Dr. FL Du
Formosan rock-monkey (Macaca cyclopis)
Thanks for Your Attention