• Molecules and Cells
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• v.24 no.3
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• pp.378-387
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• 2007

The Bcl-2 family of proteins interacts at the mitochondria to regulate apoptosis. However, the anti-apoptotic Bcl-2 and $Bcl-X_L$ are not completely localized to the mitochondria. In an attempt to generate Bcl-2 and $Bcl-X_L$ chimeras that are constitutively localized to the mitochondria, we substituted their C-terminal transmembrane tail or both the C-terminal transmembrane tail and the adjacent loop with the equivalent regions from Bak or Bax mutant (BaxS184V) as these regions determine the mitochondrial localization of Bak and Bax. The effects of these substitutions on subcellular localization and their activities were assessed following expression in HeLa and CHO K1 cells. The substitution of the C-terminal tail or the C-terminal tail and the adjacent loop of Bcl-2 with the equivalent regions from Bak or the Bax mutant resulted in its association with the mitochondria. This change in subcellular localization of Bcl-2 chimeras triggered cells to undergo apoptotic-like cell death. The localization of this Bcl-2 chimera to the mitochondria may be associated with the disruption of mitochondrial membrane potential. Unlike Bcl-2, the loop structure adjacent to the C-terminal tail in $Bcl-X_L$ is crucial for its localization. To localize the $Bcl-X_L$ chimeras to the mitochondria, the loop structure next to the C-terminal tail in $Bcl-X_L$ protein must remain intact and cannot be substituted by the loop from Bax or Bak. The chimeric $Bcl-X_L$ with both its C-terminal tail and the loop structure replaced by the equivalent regions of Bak or Bax mutant localized throughout the entire cytosol. The $Bcl-X_L$ chimeras that are targeted to the mitochondria and the wild type $Bcl-X_L$ provided same protection against cell death under several death inducing conditions.

• Korean Journal of Veterinary Research
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• v.30 no.2
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• pp.231-241
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• 1990

The present study was carried out to investigate the viability of frozen-thawed aggregated mouse embryos and to produce the chimeras. Different phenotypic embryos were obtained by mating ICR female mice with ICR or CBA male mice. The aggregated morulae were produced following aggregation of embryos at 4-, 8- and 12- to 16-cell stage. The desirable stage for the aggregation of the mouse embryos was 8- to 16-cell stage. The post-thawed in vitro survival rates of aggregated embryos in glycerol, DMSO and ethylene glycerol were 51.5, 78.6 and 69.4%, respectively. Although the higher survival was obtained in DMSO, there were no significant differences in the survival rates among the three cryoprotectants. A total of 155 frozen-thawed aggregated embryos were transferred to 11 recipient mice, 3 out of 7 offsprings were born to overt chimera. These experiments have proven that mouse chimeras can be obtained from frozen-thawed aggregated embryos.

• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.135-142
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• 2001

A novel sterategy has been established to determine the origin of the Primordial Germ Cells (PGCs) in avian embryos directly and the developmental fate of the PGCs for the application to Poultry biotechnology. Cells were removed from 1) the centre of area pellucida, 2) the outer of area pellucida and 3) the area opaca of the stage X blastoderm (Eyal-Giladi & Kochav, 1976). When the cells were removed from the centre of area pellucida, the mean number of circulating PGCs in blood was significantly decreased in the embryo at stage 15 (Hamburger & Hamilton, 1951) as compared to intact embryos. When the cells were replenished with donor cells, no reduction in the PGCs number was observed. The removal of cells at the outer of area pellucida or at the area opaca had no effect on the number of PGCs. In case, another set of the manipulated embryos were cultured ex vivo to the hatching and reared to the sexual maturity, the absence of germ cells and degeneration of seminiferous tubules was observed in resulting chickens derived from the blastoderm in which the cells were removed from the centre of the area pellucida. It was concluded that the avian Primordial Germ cells are originated at the center of area pellucida. Developmental ability of the cells to differentiate into somatic cells and germ cells in chimeras were analyzed. Somatic chimerism was detected as black feather attributed from donor cells. Molecular identification by use of female - specific DNA was performed. It was confirmed that the donor cells could be differentiated into chimeric body and erythrocytes. Donor cells retained the ability to differentiate into germline in chimeric gonads. More than 70% of the generated chimeras transmitted donor derived gametes to their offspring indicating that the cells at the center of area pellucida had the high ability to differentiate into germ cells. A molecular technique to identify germline chimerism has been developed by use of gene scan analysis. Strain specific DNA fragments were amplified by the method. It would be greatly contributed for the detection of germline chimerism. Mixed- sex chimeras which contained both male and female cells were produced to investigate the developmental fate of male and female cells in ovary and testes. The sex combinations of donor and recipient of the resulting chimeras were following 4 pairs; (1) chimeras (ZZ/ZZ) produced by a male donor (ZZ) and a male recipient (ZZ), (2) chimeras (ZW/ZW) produced by a female donor (ZW) and a female recipient (ZW), (3) chimeras (ZZ/ZW) Produce by a male donor (ZZ) and a female recipient (ZW), (4) chimeras (ZW/ZZ) produced by a female donor (ZW) and a male recipient (ZZ). It was found that genetically male avian germ cells could differentiate into functional ova and that genetically female germ cells can differentiate into functional spermatozoa in the gonad of the mixed- sex chimeras. An ability for introduction of exogenous DNA into the PGCs from stage X blastoderms were analyzed. Two reporter genes, SV-$\beta$gal and RSV-GFP, were introduced into the PGCs. Expression of bacterial/gal was improved by complexing DNA with liposome detectedcc in 75% of embryos at 3 days embryos. At the embryos incubated for 1 day, expression of the GFP was observed all the embryos. At day 3 of incubation, GFP was detected in about 70% of the manipulated embryos. In case of GFP, expression of the transgene was detected in 30 %e of the manipulated embryos. These results suggested that the cells is one of the most promising vectors for transgenesis. The established strategy should be very powerfull for application to poultry biotechnology.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.137.1-137
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• 2003

A pepper strain of Cucumber mosaic virus (Pf-CMV) induces a mild chlorotic spot symptom in zucchini squash at 9 days post-inoculation (dpi), wile Fny strain of CMV causes severe mosaic and stunting symptom at 4 dpi in this host. Pseudorecombinants were constructed between the two strains, and assessments of symptom severity were indicated that both RNA2 and RNA3 were responsible for both mildness and the slow appearance of symptom elicited by Pf-CMV in zucchini squash. With various RNA2 and RNA3 chimeras between two strains of CMV, the genetic symptom determinants of phenotype of Pf-CMV were mapped to Tyr residue at positions amino acid 267 in 2a protein and at positions amino acid 168 in 3a movement protein (MP). Chimeras changed the sequences (both changed Tyr to lie) in the codons of both amino acid 168 of 3a MP and amino acid 267 of 2a protein were resulted in the high RNA accumulation, severity of symptom, and the rapid systemic spread, suggesting that 2a replicase as well as MP is involved in virus movement. The RNA accumulation pattern of all pseudorecombinants and chimeras are identical in protoplast of zucchini squash, indicating the virus movement is responsible for the phenotypes of two CMV strains rather than virus replication.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2002.11a
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• pp.98-99
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• 2002

Collection of large number of gonadal Primordial germ cells(gPGCs) is a prerequisite factor for improving germline transmission efficacy in the aves, In this study a magnetic-activated cell sorter(MACS) was applied for improving retrieval efficacy of quail gPGCs and the migration capacity of MACS-separated gPGCs was further examined after being transplanted to recipient embryos. We also induced germline chimeras by transfer of MACS-separated quail gPGCs at the efficiency of 17.4% on average.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.175-182
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• 2021

Pluripotent stem cells could self-renew and differentiate into various cells. In particular, porcine pluripotent stem cells are useful for preclinical therapy, transgenic animals, and agricultural usage. These stem cells have naïve and primed pluripotent states. Naïve pluripotent stem cells represented by mouse embryonic stem cells form chimeras after blastocyst injection. Primed pluripotent stem cells represented by mouse epiblast stem cells and human embryonic stem cells. They could not produce chimeras after blastocyst injection. Populations of embryonic stem cells are not homogenous; therefore, reporter systems are used to clarify the status of stem cells and to isolate the cells. For this reason, studies of the OCT4 reporter system have been conducted for decades. This review will discuss the naïve and primed pluripotent states and recent progress in the development of porcine OCT4 reporter systems.

• Biomolecules & Therapeutics
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• v.8 no.3
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• pp.276-280
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• 2000

In order to identify the domains of the G$_{\alpha}$16 subunit G protein that are responsible for its activation by the Interleukin-8 receptor, a serious of chimeras between G$_{\alpha}$16 and G$_{\alpha}$11 were assessed for their abilities to be activated by these receptors. Co-expression of IL-8 receptor and chimeras in which the carboxyl-terminal regions of G$_{\alpha}$11 were replaced from 30 up to 156 amino acid residues with the corresponding regions of G$_{\alpha}$16 demonstrated that C-terminal 156 amino acid residues of the G$_{\alpha}$16 were not sufficient to confer IL-8 receptor interaction specificity. Testing of a reciprocal serious of chimeras composed of G$_{\alpha}$16 sequences at the amino terminus and G$_{\alpha}$11 sequences at the carboxyl terminals revealed that sequences extending from the amino tar- minus to amino acid 209 of G$_{\alpha}$16 were sufficient to 7ndow the chimera with 75-80% of interaction specificity for 7-8-induced activation. These results suggest th,.7t combined interactions of the C-terminal 30 amino acid residues and certain domains extending from the arts.ino terminus to amino acid 209 of Gal 6 protein may be involved in its couplings to IL-8 receptor.tain domains extending from the arts.ino terminus to amino acid 209 of Gal 6 protein may be involved in its couplings to IL-8 receptor.

• BMB Reports
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• v.45 no.3
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• pp.133-140
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• 2012

Cancers claim millions of lives each year. Early detection that can enable a higher chance of cure is of paramount importance to cancer patients. However, diagnostic tools for many forms of tumors have been lacking. Over the last few years, studies of chimeric RNAs as biomarkers have emerged. Numerous reports using bioinformatics and screening methodologies have described more than 30,000 expressed sequence tags (EST) or cDNA sequences as putative chimeric RNAs. While cancer cells have been well known to contain fusion genes derived from chromosomal translocations, rearrangements or deletions, recent studies suggest that trans-splicing in cells may be another source of chimeric RNA production. Unlike cis-splicing, trans-splicing takes place between two pre-mRNA molecules, which are in most cases derived from two different genes, generating a chimeric non-co-linear RNA. It is possible that trans-splicing occurs in normal cells at high frequencies but the resulting chimeric RNAs exist only at low levels. However the levels of certain RNA chimeras may be elevated in cancers, leading to the formation of fusion genes. In light of the fact that chimeric RNAs have been shown to be overrepresented in various tumors, studies of the mechanisms that produce chimeric RNAs and identification of signature RNA chimeras as biomarkers present an opportunity for the development of diagnoses for early tumor detection.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.71-73
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• 2001

In this study, we could improve transmission efficiency of germline chimeras by transfer of gonadal PGCs (gPGCs) cultured in vitro. Of hatched recipient chicks, 301 chickens (141 males and 160 females) were brought up to sexual maturity and these WLs (KOC) were mated with KOCs for testcross, resulting in 27 germline chimeras (15 males and 12 females) identified by black feather color of their progenies. The production efficiency of germline chimera production of experimental groups was observed (P=0.6831). The average transmission efficiency of proven germline chimeras was 0.6 ∼56.5% (15.0% on average). The transmission efficiency of experimental group which were transferred 10-days cultured gPGCs without Ficoll treatment was highest (49.7%) and that of experimental stock which transferred non-cultured gPGCs with Ficoll treatment was lowest (0.6%). The duration of in vitro culture before transferring was significantly important for the high efficiency of germline transmission. Transferring 10-days cultured gPGCs made the transmission efficiency higher rather than transferring non-cultured and 5-days cultured gPGCs, 50 times and 10 times, respectively (p<0.0001). However, Ficoll treatment for increasing the population ratio of gPGCs negatively affected the transmission efficiency and the effects of sexuality and the reciprocal interaction between treatments showed no significant differences. These findings demonstrated that the crucial factors for improving the germline transmission were the duration of in vitro culture prior to transfer. Thus, we developed the complete system for production of germline chimera using cultured gPGCs with highly improved efficiency and this system would be useful for genetic manipulation and obtaining the transgenic aves.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.2
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• pp.158-164
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• 2005

A practical approach was proposed to produce transgenic chimeric chickens using blastodermal cells (BCs). The chicken BCs were mechanically dissociated and transferred into the recipient eggs that had been exposed to 500 rads irradiation of$^{60}Co$ and windowed on the equatorial plane. Chimeric chickens were generated using two models: the crosses (MXL) from Black Minors (ii,EE,b/b) ♂${\times}$Barred Leghorns (ii,ee,B/-) ♀ as donors and White Leghorns (WL, II) as acceptors (Model 1), or the Black Heifengs (BH, ii,EE,bb) as donors and Hua-xing white (HW, II) as recipients (Model 2). The treated eggs were incubated in their original shells in normal conditions until hatching. Green fluorescent protein (GFP) gene was transferred into the BCs derived from MXL and BH via lipofectamine and the pEGFP-C1, and transfection efficiency into the BCs was examined under a fluorescent microscope. Potential transgenic chimeras were selected based on the proposed methods in this study. Using the fresh BCs, the best rate of phenotypic chimeras was 6.7% and 26.0% in model-1 groups, and model-2 groups, respectively. We also described the optimized conditions for transfection. Although 30% of the BCs transfected in vitro emitted green light under an inverted fluorescent microscope, no embryos injected with the transfected BCs expressed foreign GFP gene at 3-4 days.