Production of offspring from cloned transgenic RFP female dogs and stable generational transmission of the RFP gene

Hong, So Gun,Oh, Hyun Ju,Park, Jung Eun,Kim, Min Jung,Kim, Geon A,Park, Eun Jung,Koo, Ok Jae,Kang, Sung Keun,Jang, Goo,Lee, Byeong Chun Wiley Subscription Services, Inc., A Wiley Company 2011 Genesis Vol.49 No.11

<P><B>Abstract</B></P><P>The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild‐type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild‐type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations. genesis 49:835–840, 2011. © 2011 Wiley‐Periodicals, Inc.</P>

Growth Regulation in IGF-1 Receptor Transgenic Mice

Kim Hyun-Joo,Shin Young-Min,Chang Suk-Min,Park Chang-Sik,Jin Dong-Il The Korean Society of Animal Reproduction 2006 Reproductive & developmental biology Vol.30 No.2

To study the signaling effect of insulin-like growth factor-I(IGF-1), transgenic mice containing IGF-1 Receptor (IGF-1R) cDNA fused to metallothionein promoter were produced by DNA microinjection into the pronucleus of mouse zygote. Three founders were produced with transgenic mice containing IGF-1R gene. Transgenic mice lines contained approximately $4{\sim}20$ copies of transgenes per cell and transmission of this gene into the progeny with Mendelian manner were determined. The founder mice were mated with normal mice to produce $F_1$ mice and then $F_2$ mice. Transmission rates of IGF-1R transgene in the progeny mice were $25{\sim}60%$ in $F_1$ generation and $40{\sim}50%$ in $F_2$ generation. The mRNA expression of IGF-1R transgene in liver was analyzed using RT-PCR for IGF-1R gene in liver. When body weights of transgenic pups were measured during 4, 10 and 14 weeks after birth, IGF-1R transgenic mice grew faster than non transgenic littermates. This study indicated that growth regulation by IGF-1 signaling through IGF-1R can be elucidated using IGF-1R transgenic mice.

Growth Regulation in IGF-1 Receptor Transgenic Mice

Hyun Joo Kim,Young Min Shin,Suk Min Chang,Chang Sik Park,Dong Il Jin 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.2

To study the signaling effect of insulin-like growth factor-Ⅰ(IGF-1), transgenic mice containing IGF-1 Receptor (IGF-1R) cDNA fused to metallothionein promoter were produced by DNA microinjection into the pronucleus of mouse zygote. Three founders were produced with transgenic mice containing IGF-1R gene. Transgenic mice lines contained approximately 4~20 copies of transgenes per cell and transmission of this gene into the progeny with Mendelian manner were determined. The founder mice were mated with normal mice to produce F1 mice and then F2 mice. Transmission rates of IGF-1R transgene in the progeny mice were 25~60% in F1 generation and 40~50% in F2 generation. The mRNA expression of IGF-1R transgene in liver was analyzed using RT-PCR for IGF-1R gene in liver. When body weights of transgenic pups were measured during 4, 10 and 14 weeks after birth, IGF-1R transgenic mice grew faster than non transgenic littermates. This study indicated that growth regulation by IGF-1 signaling through IGF-1R can be elucidated using IGF-1R transgenic mice.

Transmission of Bovine β-Casein/Human Lactoferrin Fusion Gene in Transgenic Cattle

Yong-Mahn Han,Deog-Bon Koo,Jung-Sun Park,Young-Hun Kim,Kea-Joung Lee,Kyung-Kwang Lee 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & developmental biology Vol.29 No.4

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine β-casein/human lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in 50㎕ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was 20.9% (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 (8.8%) were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

Transmission of Bovine $\beta-Casein/Human$ Lactoferrin Fusion Gene in Transgenic Cattle

Han Yong-Mahn,Koo Deog-Bon,Park Jung-Sun,Kim Young-Hun,Lee Kea-Joung,Lee Kyung-Kwang The Korean Society of Animal Reproduction 2005 Reproductive & developmental biology Vol.29 No.4

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

Growth Regulation in IGF-1 Receptor Transgenic Mice

Kim, Hyun Joo,Shin, Young Min,Chang, Suk Min,Park, Chang Sik,Jin, Dong Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

To study the signaling effect of insulin-like growth factor- I(IGF-1), transgenic mice containing IGF-1 Receptor(IGF-1R) cDNA fused to metallothionein promoter were produced by DNA microinjection into the pronucleus of mouse zygote. Three founders were produced with transgenic mice containing IGF-1R gene. Transgenic mice lines contained approximately 4∼20 copies of transgenes per cell and transmission of this gene into the progeny with Mendelian manner were determined. The founder mice were mated with normal mice to produce F₁mice and then F₂mice. Transmission rates of IGF-1R transgene in the progeny mice were 25∼60% in F₁generation and 40∼50% in F₂generation. The mRNA expression of IGF-1R transgene in liver was analyzed using RT-PCR for IGF-1R gene in liver. When body weights of transgenic pups were measured during 4, 10 and 14 weeks after birth, IGF-1R transgenic mice grew faster than non transgenic littermates. This study indicated that growth regulation by IGF-1 signaling through IGF-1R can be elucidated using IGF-1R transgenic mice.

Production of Germline Transgenic Chickens Expressing High levels of Recombinant hEPO using a MoMLV-based Retrovirus Vector

Bon Chul Koo,Mo Sun Kwon,Dohyang Kim,Sanga Kim,Museog Choe,Yuhwa Nam,Teoan Kim 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10

In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of hEPO. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). One rooster was mated to wild-type hens to produce 748 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were seven G1 transgenic offspring, corresponding to a 0.9% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from three G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood samples taken from G1 transgenic chickens resulted in 4,150 ~ 10,823 IU/㎖ (34.6 ~ 90.2 ㎍/㎖) of hEPO in the blood. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. Red blood cell numbers were more than three-fold higher in the transgenic chickens compared to the non-transgenic chickens. Successful germline transmission of the transgene was also confirmed in G2 transgenic chicks produced from crossing G1 transgenic roosters with non-transgenic hens. We confirmed that 13 transgenic chicks of 45 G2 progeny, corresponding to a 28.9% germline transmission rate. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

Expression of Recombinant hEPO in the Egg White of Transgenic Chickens

Bon Chul Koo,Mo Sun Kwon,Dohyang Kim,Yuhwa Nam,Teoan Kim 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

In this study, we report successful expression of recombinant human erythropoietin (hEPO) in the egg white of transgenic hens using a feline immunodeficiency virus (FIV)- based lentiviral vector as an exogenous gene deliverer. hEPO is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. FIV vectors permit high levels of transgene expression in quails and chickens. We constructed a FIV vector containing a hEPO cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. Out of 208 injected eggs, 10 chicks were hatched after 21 days of incubation, and one of the G0 hatched chicken expressed the vector-encoded hEPO gene in sperm. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. One rooster was mated to wild-type hens to produce 518 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were four G1 transgenic offspring, corresponding to a 0.77% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from four G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood and egg white samples taken from G1 transgenic chickens resulted in 4,810~6,600 IU/mL (40.1~55.0 ㎍/mL) of hEPO in egg white, whereas 18~25 mIU/mL (0.15~0.2 ng/mL) in serum. The biological activity of the recombinant hEPO in egg white was comparable to its commercially available counterpart. We conclude that successful expression of recombinant hEPO in the egg white of transgenic chickens implies an important step towards efficient production of human cytokine from the transgenic animal bioreactor.

Expression of the S glycoprotein of transmissible gastroenteritis virus (TGEV) in transgenic potato and its immunogenicity in mice

Ahn, Dong-Joo,Youm, Jung Won,Kim, Suk Weon,Yoon, Won Kee,Kim, Hyoung Chin,Hur, Tai-Young,Joung, Young Hee,Jeon, Jae-Heung,Kim, Hyun Soon The Korean Society of Veterinary Science 2013 大韓獸醫學會誌 Vol.53 No.4

Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-$S_{0.7}$) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-$S_{0.7}$ gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-$S_{0.7}$ protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-$S_{0.7}$ antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

형질전환 생쥐의 후대에서 인간 Interleukin-10 유전자의 안정적 전이와 지속적인 발현

정진우,구덕본,한용만,이경광 한국동물생명공학회(구 한국동물번식학회) 2004 Reproductive & developmental biology Vol.28 No.3

형질전환 동물의 유선에서 특이적으로 발현되도록 고안된 pBIL-10 발현 벡터를 이용하여 인간 IL-10 유전자가 삽입되어 한 계통으로 확립된 형질전환 생쥐에서 이 유전자가 장기 세대까지 안정적으로 전이되고, 또한 발현 수준도 지속적으로 유지되는지를 조사하였다. 이를 위해 제 8 세대의 수컷 hIL-10 형질전환 생쥐를 실험에 공시하였고, 제 15 세대까지의 전이율과 hIL-10 유전자의 발현 수준을 분석하였다, 제 8 세대 생쥐의 계대 번식에 의한 자손 중 50.9±5.8%가 형질전환 생쥐로 판명되었다. 또한 제 9 세대에서 외래 유전자의 전이율은 66.0±20.1%이렀고, 제 10 세대에서 외래 유전자의 전이율은 61.5±16.7%이었고, 제 11 세대에서 외래 유전자의 전이율은 41.1±8.4%이었고, 제 12 세대에서 외래 유전자의 전이율은 40.7±20.3%이었고, 제 13 세대에서 외래 유전자의 전이율은 61.3±10.8%이었고, 제 14 세대에서 외래 유전자의 전이율은 49.2±18.8%이었고, 제 15 세대에서 외래 유전자의 전이율은 43.8±25.9%이었다. 이러한 결과로 hIL-10 형질전환 생쥐는 그 외래유전자의 유전적 손상이 없이 장기 세대까지 안정적으로 전이되는 것으로 판다된다. 제 9 세대의 암컷 형질전환 생쥐로부터 유즙내 인간 hIL-10의 발현 수준을 분석하였을 때, 그 농도는 평균 3.6± 1.2 mg/ml의 수준에서 측정되었다. 제 10세대에서는 유즙내 인간 hIL-10의 발현 수준을 분석하였을 때, 그 농도는 평균 4.2±0.9 mg/ml의 수준에서 측정되었고, 제 11세대에서는 유즙내 인간 hIL-10의 발현 수준을 분석하였을 때, 그 농도는 평균 5.7± 1.5 mg/ml의 수준에서 측정되었고, 제 12세대에서는 유즙내 인간 hIL-10의 발현 수준을 분석하였을 때, 그 농도는 평균 6.3±3.5 mg/ml의 수준으로 측정되었고, 제 13세대에서는 유즙내 인간 hIL-10의 발현 수준을 분석하였을 때, 그 농도는 평균 6.8±4.5 mg/ml의 수준으로 측정되었고, 제 14세대에서는 유즙내 인간 hIL-10의 발현 수준을 분석하였을 때, 그 농도는 평균 6.8±3.1 mg/ml의 수준으로 측정되었다. 이러한 수준은 제 1 세대의 것보다 높은 결과로 형질전환 생쥐에서 인간 IL-10 유전자의 발현은 최소한 15 세대까지 지속적으로 유지된다는 것을 알 수 있었으며, 장기 세대까지도 발현수준이 유지될 것으로 판단된다. 이러한 연구결과는 계통으로 확립된 형질전환 동물에 부여된 새로운 유전형질은 지속적으로 후대로 유전될 수 있음을 제시한다. The transgenic mice carrying human Interleukin-10 (hIL-10) gene in conjunction with bovine (3 -casein promoter express hIL-10 in milk during lactation. In this study, stability of germ line transmission and expression of hIL-10 transgene integrated into host chromosome were monitored up to generation F8 of transgenic mice. When male mouse of generation F8 was crossbred with normal females, approximately half of offspring (50.9±5.8%) were identified as transgenic mice. Generation F9 to F15 mice also showed similar transmission rates (66.0±20.1%, 61.5±16.7%, 41.1±8.4%, 40.7±20.3%, 61.3±10.8%, 49.2±18.8% and 43.8±25.9%, respectively), implying that hIL-10 transgene can be transmitted stably up to long term generation in the transgenic mice. Expression levels of human IL-10 from milk of generation F9 to F14 mice were 3.6± 1.2 mg/ml, 4.2±0.9 mg/ml, 5.7±1.5 mg/ml, 6.3±3.5 mg/ml, 6.8±4.5 mg/ml and 6.8±3.1 mg/ml, respectively, which was showed high-level expression compared with that of generation F1 (1.6 mg/ml) mice. In conclusion, our results suggest that transgenic mice can be continuously passed their transgenes to the progeny through the breeding program with the same productivity of human IL-10 protein in their milk.