Inhibition of zebrafish fgf8 Pre-mRNA splicing with morpholino oligos:: A quantifiable method for gene knockdown
Inhibition of zebrafish fgf8 Pre-mRNA splicing with morpholino oligos:: A quantifiable method for gene knockdown
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DOI：
10.1002/gene.1053
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发表时间：
2001-07-01
期刊：
影响因子：
1.5
通讯作者：
Kimmel, CB
中科院分区：
文献类型：
--
作者：
Draper, BW;Morcos, PA;Kimmel, CB
反义吗啉代寡核苷酸（MO）已成功用于斑马鱼和非洲爪蟾，通过基因特异性抑制mRNA翻译来敲低基因功能（Ekker，2000）。除了它们阻断胞质过程的能力之外，MO还可以进入细胞核（Partial等人，1996），并已显示是哺乳动物组织培养细胞系中前mRNA剪接的有效抑制剂（Schmajuk等，1999年）。我们在这里表明，MO有效地阻止前mRNA在斑马鱼胚胎中的剪接。剪接阻断MO具有以下优点：基因敲低的功效可以在不使用抗体的情况下定量，并且它们特异性靶向合子而不是母体转录物。我们靶向fgf 8基因（Furthauer等人，1997; Reifers等人，1998年）与拼接阻断MO。ENU诱导的fgf 8突变，acerebellar（ace;在此称为fgf 8it 282），先前已被描述（Reifers等人，1998年）。fgf 8it 282是导致产生异常剪接的mRNA的剪接供体突变（Reifers等人，1998年）。因此，使用MO阻断相同的剪接事件应导致与fgf 8it 282相似的表型。我们确定了fgf 8外显子/内含子结构，并设计了两个与外显子2和外显子3剪接供体位点互补的25-mer MO（分别命名为E2 I2和E3 I3;图1a）。两种MO均跨越外显子/内含子连接，包括剪接供体共有序列的最保守残基（图1图例）。我们首先询问剪接位点靶向MO是否可以改变fgf 8 mRNA的剪接。使用逆转录聚合酶链反应，我们发现将E3 I3 MO注射到早期斑马鱼胚胎中导致产生两种异常剪接的信息，我们称之为变体1和2（图1a，B）。相应cDNA的序列分析显示，变体1是由于排除外显子3而产生的，而变体2是由于使用位于32个碱基5！正常外显子3剪接供体（图1c）。使用隐蔽剪接供体在新剪接点处产生提前终止密码子（图1c）。此外，我们发现注射的剪接位点靶向MO仅改变合子表达的mRNA的结构，而不是母体提供的mRNA的结构，如预期的那样（图2d）。因此，E3 I3可以
Antisense morpholino oligonucleotides (MO) have been used successfully in zebrafish and Xenopus to knock down gene function by gene-specific inhibition of mRNA translation (Ekker, 2000). In addition to their ability to block cytosolic processes, MO can enter the nucleus (Partridge et al., 1996) and have been shown to be effective inhibitors of pre-mRNA splicing in mammalian tissue-culture cell lines (Schmajuk et al., 1999). We show here that MO efficiently block pre-mRNA splicing in zebrafish embryos. Splice-blocking MO have the advantages that the efficacy of gene knockdown can be quantified without the use of antibodies, and that they specifically target zygotic, and not maternal, transcripts. We targeted the fgf8 gene (Furthauer et al., 1997; Reifers et al., 1998) with splice-blocking MO. An ENU induced mutation in fgf8, acerebellar (ace; referred to here as fgf8it282), has previously been described (Reifers et al., 1998). fgf8it282 is a splice donor mutation that results in the production of an aberrantly spliced mRNA (Reifers et al., 1998). Hence, blocking the same splicing event using MO should result in a phenotype similar to fgf8it282.We determined the fgf8 exon/intron structure and designed two 25-mer MO complementary to the exon 2 and exon 3 splice donor sites (designated E2I2 and E3I3, respectively; Fig. 1a). Both MO span the exon/intron junction, including the most conserved residues of the splice donor consensus sequence (Fig. 1 legend). We first asked whether splice site–targeted MO can alter splicing of fgf8 mRNA. Using reverse transcriptase polymerase chain reaction, we found that injection of the E3I3 MO into early zebrafish embryos results in the production of two aberrantly spliced messages we term variant 1 and 2 (Fig. 1a, b). Sequence analysis of the corresponding cDNAs revealed that variant 1 results from excluding exon 3, whereas variant 2 results from the use of a cryptic splice donor located 32 bases 5! of the normal exon 3 splice donor (Fig. 1c). Use of the cryptic splice donor creates a premature termination codon at the novel splice junction (Fig. 1c). Additionally, we found that injected splice site–targeted MO only alter the structure of zygotically expressed, but not maternally supplied, mRNA, as expected (Fig. 2d). Thus, E3I3 can