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Lack of Cul4b, an E3 ubiquitin ligase component, leads to embryonic lethality and abnormal placental development.

Jiang B, Zhao W, Yuan J, Qian Y, Sun W, Zou Y, Guo C, Chen B, Shao C, Gong Y - PLoS ONE (2012)

Bottom Line: Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay.The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay.As in human CUL4B heterozygotes, Cul4b cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Teratology, Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

ABSTRACT
Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B mutation, Cul4b mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse.

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Generation of Cul4b flox mice.(A) Strategy for generation of Cul4b floxed targeting vector. On the top was shown the wild-type allele of Cul4b gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. BamHI (labeled B) and XbaI (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. BamHI digested DNA was hybridized with 5′ probe and XbaI digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of Cul4b gene from the brain tissue of brain-specific knockout mice (Cul4bflox/Y;Nesin-Cre). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of Cul4b mRNA isolated from brain tissues of wild-type males (Cul4b+/Y;Nestin-Cre+/−), wild-type females (Cul4b+/+;Nestin-Cre+/−), heterozygous females (Cul4b+/flox;Nestin-Cre+/−), and conditional knock-out males (Cul4bflox/Y;Nestin-Cre+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.
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pone-0037070-g001: Generation of Cul4b flox mice.(A) Strategy for generation of Cul4b floxed targeting vector. On the top was shown the wild-type allele of Cul4b gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. BamHI (labeled B) and XbaI (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. BamHI digested DNA was hybridized with 5′ probe and XbaI digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of Cul4b gene from the brain tissue of brain-specific knockout mice (Cul4bflox/Y;Nesin-Cre). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of Cul4b mRNA isolated from brain tissues of wild-type males (Cul4b+/Y;Nestin-Cre+/−), wild-type females (Cul4b+/+;Nestin-Cre+/−), heterozygous females (Cul4b+/flox;Nestin-Cre+/−), and conditional knock-out males (Cul4bflox/Y;Nestin-Cre+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.

Mentions: Because CUL4B-deficient cells are strongly selected against [17], we envisaged that it might be difficult to generate Cul4b-deficient embryonic stem (ES) cells via conventional knockout technology. We therefore used the Cre/loxP strategy to generate Cul4b floxed ES cells. First, a Cul4b floxed targeting vector was constructed (Fig. 1A). In this vector, exons 3–5 were floxed by two loxP sites. A neo gene flanked by two FRT sites and a TK gene were also insert into intron 5 and vector backbone, respectively, for ES cells selection.


Lack of Cul4b, an E3 ubiquitin ligase component, leads to embryonic lethality and abnormal placental development.

Jiang B, Zhao W, Yuan J, Qian Y, Sun W, Zou Y, Guo C, Chen B, Shao C, Gong Y - PLoS ONE (2012)

Generation of Cul4b flox mice.(A) Strategy for generation of Cul4b floxed targeting vector. On the top was shown the wild-type allele of Cul4b gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. BamHI (labeled B) and XbaI (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. BamHI digested DNA was hybridized with 5′ probe and XbaI digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of Cul4b gene from the brain tissue of brain-specific knockout mice (Cul4bflox/Y;Nesin-Cre). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of Cul4b mRNA isolated from brain tissues of wild-type males (Cul4b+/Y;Nestin-Cre+/−), wild-type females (Cul4b+/+;Nestin-Cre+/−), heterozygous females (Cul4b+/flox;Nestin-Cre+/−), and conditional knock-out males (Cul4bflox/Y;Nestin-Cre+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351389&req=5

pone-0037070-g001: Generation of Cul4b flox mice.(A) Strategy for generation of Cul4b floxed targeting vector. On the top was shown the wild-type allele of Cul4b gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. BamHI (labeled B) and XbaI (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. BamHI digested DNA was hybridized with 5′ probe and XbaI digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of Cul4b gene from the brain tissue of brain-specific knockout mice (Cul4bflox/Y;Nesin-Cre). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of Cul4b mRNA isolated from brain tissues of wild-type males (Cul4b+/Y;Nestin-Cre+/−), wild-type females (Cul4b+/+;Nestin-Cre+/−), heterozygous females (Cul4b+/flox;Nestin-Cre+/−), and conditional knock-out males (Cul4bflox/Y;Nestin-Cre+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.
Mentions: Because CUL4B-deficient cells are strongly selected against [17], we envisaged that it might be difficult to generate Cul4b-deficient embryonic stem (ES) cells via conventional knockout technology. We therefore used the Cre/loxP strategy to generate Cul4b floxed ES cells. First, a Cul4b floxed targeting vector was constructed (Fig. 1A). In this vector, exons 3–5 were floxed by two loxP sites. A neo gene flanked by two FRT sites and a TK gene were also insert into intron 5 and vector backbone, respectively, for ES cells selection.

Bottom Line: Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay.The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay.As in human CUL4B heterozygotes, Cul4b cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Teratology, Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

ABSTRACT
Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B mutation, Cul4b mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse.

Show MeSH
Related in: MedlinePlus