Molecular cloning and functional analysis of cDNA encoding a rat leukemia inhibitory factor: towards generation of pluripotent rat embryonic stem cells

Takahama, Yasushi; Ochiya, Takahiro; Sasaki, Hiroki; Baba-Toriyama, Hiroyasu; Konishi, Hideyuki; Nakano, Hirofumi; Terada, Masaaki

doi:10.1038/sj.onc.1201826

Cited by 19 publications

(11 citation statements)

References 21 publications

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“…was up-regulated after stimulation with rat LIF (27) even in the YPAC medium. Thus, it seems possible to improve the culture condition further by the administration of rat LIF.…”

Section: Discussionmentioning

confidence: 93%

Generation of genetically modified rats from embryonic stem cells

Kawamata¹,

Ochiya²

2010

Proc. Natl. Acad. Sci. U.S.A.

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At present, genetically modified rats have not been generated from ES cells because stable ES cells and a suitable injection method are not available. To monitor the pluripotency of rat ES cells, we generated Oct4-Venus transgenic (Tg) rats via a conventional method, in which Venus is expressed by the Oct4 promoter/enhancer. This monitoring system enabled us to define a significant condition of culture to establish authentic rat ES cells based on a combination of 20% FBS and cell signaling inhibitors for Rhoassociated kinase, mitogen-activated protein kinase, TGF-β, and glycogen synthase kinase-3. The rat ES cells expressed ES cell markers such as Oct4, Nanog, Sox2, and Rex1 and retained a normal karyotype. Embryoid bodies and teratomas were also produced from the rat ES cells. All six ES cell lines derived from three different rat strains successfully achieved germline transmission, which strongly depended on the presence of the inhibitors during the injection process. Most importantly, high-quality Tg rats possessing a correct transgene expression pattern were successfully generated via the selection of gene-manipulated ES cell clones through germline transmission. Our rat ES cells should be sufficiently able to receive gene targeting as well as Tg manipulation, thus providing valuable animal models for the study of human diseases.genetic engineering | rat | embryonic stem cells T he laboratory rat was the earliest mammalian species domesticated for scientific research and has been used as an animal model in physiology, toxicology, nutrition, behavior, immunology, and neoplasia for over 150 y (1). Despite this history, rats lag far behind mice in functional genetic studies and the generation of knockout animal models reflecting human diseases because of the absence of germline-competent rat ES cells, which are vital in a reverse genetics approach (2, 3). Recently, gene-targeting rats were created by the zinc finger nuclease strategy (4). However, the system is not available for most researchers because a special technique is required to make algorithm-based sequence-specific DNA nucleases. Thus, establishment of rat ES cells has been desired to produce gene-targeting rats, such as mutant mice, routinely.Although we established rat ES cell lines with chimeric contribution, none could complete germline transmission (5). Soon after our report, other groups succeeded in establishing rat ES cells with germline transmission by using 2i, mitogen-activated protein kinase (MEK) inhibitor PD0325901, and glycogen synthase kinase-3 (GSK3) inhibitor CHIR99021 (6, 7). The 2i is widely used in the establishment of ES cells or induced pluripotent stem (iPS) cells in mice (8, 9), rats (6, 7, 10), and humans (10). Thus, the inhibition of MEK and GSK3 has been thought to maintain a ground state of pluripotency in various species. Rat iPS cells with chimeric contribution were established by using an inhibitor of type 1 TGF-β receptor Alk5 (A-83-01) with the 2i, although germline transmission was not accomplished (10). Furthermore...

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“…was up-regulated after stimulation with rat LIF (27) even in the YPAC medium. Thus, it seems possible to improve the culture condition further by the administration of rat LIF.…”

Section: Discussionmentioning

confidence: 93%

Generation of genetically modified rats from embryonic stem cells

Kawamata¹,

Ochiya²

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…In some experiments on rat cells, rat LIF [27] was used, with comparable results. EHS cell attachment matrix (Promega, Madison, WI) was added at a concentration of 140 l/ ml.…”

Section: Mediummentioning

confidence: 98%

Rapid Loss of Oct-4 and Pluripotency in Cultured Rodent Blastocysts and Derivative Cell Lines1

Buehr¹,

Nichols²,

Stenhouse³

et al. 2003

Biology of Reproduction

138

101

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The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass (ICM) and derivative embryonic stem (ES) cells. We analyzed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat preimplantation embryos. Oct-4 was rapidly lost in primary outgrowths of the majority of cultured embryos prior to any evidence of morphological differentiation. Oct-4 persisted in only a minority of strain 129 cultures, which can go on to give ES cells. We used transgenic rats in which the dual reporter/selection marker beta-geo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. Ablation of all cells occurred, consistent with complete downregulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could be derived readily from rat blastocysts and ICMs. However, these cells did not express significant Oct-4 and, although capable of differentiating into extraembryonic cell types, appeared incapable of producing fetal germ layer derivatives. Downregulation of Oct-4 appears to be a limiting factor in attempts to derive pluripotent cell lines from preimplantation embryos.

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“…Transverse sections (14 m) were cut in a cryostat (Microm, Heidelberg, Germany), thawed onto Probe-On slides (Fisher Scientific, Pittsburg, PA), and stored in black, sealed boxes at -70°C until use. The synthetic oligonucleotides were complementary to rat mRNA encoding GAP-43 (nucleotides 70 -117; Karns et al, 1987), GAPDH (nucleotides 860 -902; Fort et al, 1985), BDNF (nucleotides 558 -599 and 645-694; Maisonpierre et al, 1991), NT-3 (nucleotides 286 -327 and 538 -579;Ernfors et al, 1990), LIF (nucleotides 492-539; Takahama et al, 1998), CNTF (nucleotides 286 -327; (Stockli et al, 1989), GDNF (nucleotides 271-312 and 540 -589;Lin et al, 1993), the tyrosine kinase domain of full-length trkB (nucleotides 2,576 -2,617; Middlemas et al, 1991), trkC (nucleotides 1,189 -1,239;Merlio et al, 1992; nonselective for catalytic and noncatalytic isoforms), gp130 (nucleotides 987-1,034 and 1,908 -1,955; , CNTFR␣ (nucleotides 467-514; Ip et al, 1993a), LIFR (nucleotides 3,009 -3,056; Aikawa et al, 1997), GFR␣-1 (nucleotides 341-388; Jing et al, 1996), and c-RET (nucleotides 260 -307; Moreau et al, 1998). The sequences of the probes were run in a GenBank database search to exclude significant homology with unrelated genes.…”

Section: In Situ Hybridization Histochemistrymentioning

confidence: 99%

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat

et al. 2000

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After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.

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Molecular cloning and functional analysis of cDNA encoding a rat leukemia inhibitory factor: towards generation of pluripotent rat embryonic stem cells

Cited by 19 publications

References 21 publications

Generation of genetically modified rats from embryonic stem cells

Generation of genetically modified rats from embryonic stem cells

Rapid Loss of Oct-4 and Pluripotency in Cultured Rodent Blastocysts and Derivative Cell Lines1

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat

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