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Newly developed TGF-β2 knock down transgenic mouse lines express TGF-β2 differently and its distribution in multiple tissues varies.

Xiyang YB, Wang F, Qian BJ, You L, Lu BT, Zhang W, Quan XZ, Ge WP, Liu S, Zhang LF, Wang TH - BMC Biochem. (2013)

Bottom Line: Transforming growth factor-betas (TGF-βs), including beta2 (TGF-β2), constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation and tissue remodeling.TGF-β2 is thought to play important roles in multiple developmental processes and neuron survival.However, before we carried out these investigations, a TGF-β2 gene down-regulated transgenic animal model was needed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming 650500, Yunnan, China.

ABSTRACT

Background: Transforming growth factor-betas (TGF-βs), including beta2 (TGF-β2), constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation and tissue remodeling. TGF-β2 is thought to play important roles in multiple developmental processes and neuron survival. However, before we carried out these investigations, a TGF-β2 gene down-regulated transgenic animal model was needed. In the present study, expressional silencing TGF-β2 was achieved by select predesigning interference short hairpin RNAs (shRNAs) targeting mouse TGF-β2 genes.

Results: Four homozygous transgenic offspring were generated by genetic manipulation and the protein expressions of TGF-β2 were detected in different tissues of these mice. The transgenic mice were designated as Founder 66, Founder 16, Founder 53 and Founder 41. The rates of TGF-β2 down-expression in different transgenic mice were evaluated. The present study showed that different TGF-β2 expressions were detected in multiple tissues and protein levels of TGF-β2 decreased at different rates relative to that of wild type mice. The expressions of TGF-β2 proteins in transgenic mice (Founder 66) reduced most by 52%.

Conclusions: The present study generated transgenic mice with TGF-β2 down-regulated, which established mice model for systemic exploring the possible roles of TGF-β2 in vivo in different pathology conditions.

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Recombination plasmid for Tg mice with TGF-β2 down-regulation. A: showed the schedules of recombination plasmid for pcDNA6.2-GW/EmGFP-miR of TGF-β2 gene silence, which composed with 5699 nucleotides. The 293T cells were transfected with the transgenic vectors (pcDNA3.1 (+) of pcDNA6.2-GW/EmGFP-miR-TGF-β1). RT-PCR was employed to evaluate the effects of PDGF-BB down-regulation transformants. B: showed the amplification plot of RT-PCR. Red arrows showed the selected cell lines as they had the lowest levels. Black arrow indicated the control ones. C: shows the represented bands of semi-quantity PCR products electrophoresed in 1% agarose gel stained with EB. Lane 1: DL2000 DNA Marker (from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp respectively); Lane 2–4: 293T cells transfected with silence expression vector for TGF-β2 gene (lane 3: NO.21). Arrows in Figure 5C revealed the target transformants (NO.21) for TGF-β2 expressional silence as they had the lowest levels.
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Figure 5: Recombination plasmid for Tg mice with TGF-β2 down-regulation. A: showed the schedules of recombination plasmid for pcDNA6.2-GW/EmGFP-miR of TGF-β2 gene silence, which composed with 5699 nucleotides. The 293T cells were transfected with the transgenic vectors (pcDNA3.1 (+) of pcDNA6.2-GW/EmGFP-miR-TGF-β1). RT-PCR was employed to evaluate the effects of PDGF-BB down-regulation transformants. B: showed the amplification plot of RT-PCR. Red arrows showed the selected cell lines as they had the lowest levels. Black arrow indicated the control ones. C: shows the represented bands of semi-quantity PCR products electrophoresed in 1% agarose gel stained with EB. Lane 1: DL2000 DNA Marker (from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp respectively); Lane 2–4: 293T cells transfected with silence expression vector for TGF-β2 gene (lane 3: NO.21). Arrows in Figure 5C revealed the target transformants (NO.21) for TGF-β2 expressional silence as they had the lowest levels.

Mentions: TGF-β2 knock down (TGF-β2-kd) transgenic (Tg) mice with C57BL/6J genetic background were produced by our collaborators in The Institute of Laboratory Animal Science (Chinese Academy of Medical Sciences & Comparative Medicine Centre, Peking Union Medical College, Beijing, China). The generation of the transgenic mice was described as follows. Briefly, at least three silence expression sites of TGF-β2 were designed by software supplied by Invitrogen Company, USA. Then we selected predesigned short hairpin RNA (shRNAs) that target mouse TGF-β2 gene (Mus musculus, GeneID: 21808). The reconstruction plasmid was designed (Figure 5A) and purchased from Invitrogen Company. The constructed recombinant plasmid was transferred into 293T cells. The transformants were screened and identified by polymers chain reaction (PCR) detections and restriction analysis (Figure 5B, C and D).


Newly developed TGF-β2 knock down transgenic mouse lines express TGF-β2 differently and its distribution in multiple tissues varies.

Xiyang YB, Wang F, Qian BJ, You L, Lu BT, Zhang W, Quan XZ, Ge WP, Liu S, Zhang LF, Wang TH - BMC Biochem. (2013)

Recombination plasmid for Tg mice with TGF-β2 down-regulation. A: showed the schedules of recombination plasmid for pcDNA6.2-GW/EmGFP-miR of TGF-β2 gene silence, which composed with 5699 nucleotides. The 293T cells were transfected with the transgenic vectors (pcDNA3.1 (+) of pcDNA6.2-GW/EmGFP-miR-TGF-β1). RT-PCR was employed to evaluate the effects of PDGF-BB down-regulation transformants. B: showed the amplification plot of RT-PCR. Red arrows showed the selected cell lines as they had the lowest levels. Black arrow indicated the control ones. C: shows the represented bands of semi-quantity PCR products electrophoresed in 1% agarose gel stained with EB. Lane 1: DL2000 DNA Marker (from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp respectively); Lane 2–4: 293T cells transfected with silence expression vector for TGF-β2 gene (lane 3: NO.21). Arrows in Figure 5C revealed the target transformants (NO.21) for TGF-β2 expressional silence as they had the lowest levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750643&req=5

Figure 5: Recombination plasmid for Tg mice with TGF-β2 down-regulation. A: showed the schedules of recombination plasmid for pcDNA6.2-GW/EmGFP-miR of TGF-β2 gene silence, which composed with 5699 nucleotides. The 293T cells were transfected with the transgenic vectors (pcDNA3.1 (+) of pcDNA6.2-GW/EmGFP-miR-TGF-β1). RT-PCR was employed to evaluate the effects of PDGF-BB down-regulation transformants. B: showed the amplification plot of RT-PCR. Red arrows showed the selected cell lines as they had the lowest levels. Black arrow indicated the control ones. C: shows the represented bands of semi-quantity PCR products electrophoresed in 1% agarose gel stained with EB. Lane 1: DL2000 DNA Marker (from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp respectively); Lane 2–4: 293T cells transfected with silence expression vector for TGF-β2 gene (lane 3: NO.21). Arrows in Figure 5C revealed the target transformants (NO.21) for TGF-β2 expressional silence as they had the lowest levels.
Mentions: TGF-β2 knock down (TGF-β2-kd) transgenic (Tg) mice with C57BL/6J genetic background were produced by our collaborators in The Institute of Laboratory Animal Science (Chinese Academy of Medical Sciences & Comparative Medicine Centre, Peking Union Medical College, Beijing, China). The generation of the transgenic mice was described as follows. Briefly, at least three silence expression sites of TGF-β2 were designed by software supplied by Invitrogen Company, USA. Then we selected predesigned short hairpin RNA (shRNAs) that target mouse TGF-β2 gene (Mus musculus, GeneID: 21808). The reconstruction plasmid was designed (Figure 5A) and purchased from Invitrogen Company. The constructed recombinant plasmid was transferred into 293T cells. The transformants were screened and identified by polymers chain reaction (PCR) detections and restriction analysis (Figure 5B, C and D).

Bottom Line: Transforming growth factor-betas (TGF-βs), including beta2 (TGF-β2), constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation and tissue remodeling.TGF-β2 is thought to play important roles in multiple developmental processes and neuron survival.However, before we carried out these investigations, a TGF-β2 gene down-regulated transgenic animal model was needed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming 650500, Yunnan, China.

ABSTRACT

Background: Transforming growth factor-betas (TGF-βs), including beta2 (TGF-β2), constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation and tissue remodeling. TGF-β2 is thought to play important roles in multiple developmental processes and neuron survival. However, before we carried out these investigations, a TGF-β2 gene down-regulated transgenic animal model was needed. In the present study, expressional silencing TGF-β2 was achieved by select predesigning interference short hairpin RNAs (shRNAs) targeting mouse TGF-β2 genes.

Results: Four homozygous transgenic offspring were generated by genetic manipulation and the protein expressions of TGF-β2 were detected in different tissues of these mice. The transgenic mice were designated as Founder 66, Founder 16, Founder 53 and Founder 41. The rates of TGF-β2 down-expression in different transgenic mice were evaluated. The present study showed that different TGF-β2 expressions were detected in multiple tissues and protein levels of TGF-β2 decreased at different rates relative to that of wild type mice. The expressions of TGF-β2 proteins in transgenic mice (Founder 66) reduced most by 52%.

Conclusions: The present study generated transgenic mice with TGF-β2 down-regulated, which established mice model for systemic exploring the possible roles of TGF-β2 in vivo in different pathology conditions.

Show MeSH
Related in: MedlinePlus