Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model.
Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model.
复制标题
DOI：
10.1016/j.matbio.2011.12.004
复制
发表时间：
2012-04
期刊：
影响因子：
6.9
通讯作者：
McAlinden, Audrey
中科院分区：
文献类型：
--
作者：
Lewis, Renate;Ravindran, Soumya;Wirthlin, Louisa;Traeger, Geoffrey;Fernandes, Russell J.;McAlinden, Audrey
关键词：
本研究描述了一种基因敲入小鼠模型的产生，以解决II型前胶原（Col2a1）选择性剪接在骨骼发育和维护中的作用。Col2a1前体mRNA的选择性剪接是仅发生在软骨形成组织中的发育调节事件。正常情况下，软骨祖细胞主要合成含有外显子2的mRNA同种型（IIA和IID型），而缺乏外显子2的Col2a1 mRNA（IIB型）是分化的软骨细胞产生的主要同种型。另一种异构体IIC也已被鉴定，其含有截短的外显子2并且不翻译成蛋白质。这种IIA/IID至IIB剪接开关的生物学意义尚不清楚。利用剪接位点靶向敲入方法，产生4个核苷酸突变以将Col2a1外显子2的5个剪接位点从弱的非共有序列转化为强的共有剪接位点。这导致仅IIA mRNA同种型的明显表达，通过体外剪接含有5 ′剪接位点突变的II型前胶原小基因证实。为了在体内测试剪接位点靶向方法，产生了经工程改造以保留IIA外显子2（Col2a1 + ex 2）的纯合子小鼠。来自纯合子小鼠后肢骺软骨的软骨细胞显示在分析的所有出生前和出生后发育阶段（E12.5、E16.5、P0、P3、P7、P14、P28和P70）仅表达IIA mRNA和蛋白。正如预期的那样，IIB型前胶原是在所有出生后时间点在野生型软骨中产生的主要同种型。Col2a1 + ex2纯合子小鼠是活的，看起来健康，迄今为止没有显示明显的表型。然而，研究目前正在进行调查的生物学后果的持续表达的外显子2编码的保守的富含半胱氨酸的结构域在出生后的骨骼组织。
The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5 splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5′ splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1+ex2) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1+ex2 homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.
登录
查看更多内容
DOI：
10.1002/bdrc.20004
发表时间：
2004-03-01
期刊：
Birth defects research. Part C, Embryo today : reviews
影响因子：
--
作者：
McAlinden, Audrey;Havlioglu, Necat;Sandell, Linda J
通讯作者：
Sandell, Linda J
影响因子：
4.8
作者：
Fernandes, RJ;Hirohata, S;Apte, SS
通讯作者：
Apte, SS
影响因子：
4.8
作者：
Fukui, N;McAlinden, A;Sandell, LJ
通讯作者：
Sandell, LJ
影响因子：
4.8
作者：
McAlinden, Audrey;Liang, Li;Sandell, Linda J.
通讯作者：
Sandell, Linda J.
DOI：
10.1186/ar380
发表时间：
2002
期刊：
Arthritis research
影响因子：
--
作者：
Eyre D
通讯作者：
Eyre D