条件性基因敲除(conditional knock out)与Cre/loxP、Flp/FRT重组系统

        要做这方面的实验，查了些条件性基因敲除与Cre/loxP、Flp/FRT重组系统的资料，大概总结一下。

    经典基因敲除有时因某些基因对胚胎发育的影响而无法正常分娩，或实验动物因出生后严重的生理缺陷而过早死亡，或不能产生后代而不能获得纯合子动物模型。这些，可以通过条件性基因敲出或敲入（conditional gene knock out or knock in）而避免,即在特定的组织细胞或细胞发育的特定阶段敲除或引入某一特定基因，对研究特定基因在特定组织内（及特定的时间）的功能有着重要意义。

    目前常用Cre/loxP、Flp/FRT重组系统。

    利用Cre／LoxP 和来自酵母的Flp—FRT系统可以研究特定组织器官或特定细胞中靶基因灭活所导致的表型。通过常规基因打靶在基因组的靶位点上装上两个同向排列的1oxP，并以此两侧装接上loxP 的(“loxP floxed”)ES 细胞产生“loxPfloxed”小鼠,然后，通过将“loxP floxed”小鼠与Cre 转基因鼠杂交(也可以其他方式向小鼠中引入Cre 重组酶)，产生靶基因发生特定方式(如特定的组织特异性)修饰的条件性突变小鼠。在“loxP floxed”小鼠，虽然靶基因的两侧已各装上了一个loxP，但靶基因并没有发生其他的变化，故“1oxP floxed”小鼠表型仍同野生型的一样。但当它与Cre 转基因小鼠杂交时，产生的子代中将同时带有“loxP floxed”靶基因和Cre 基因。Cre 基因表达产生的Cre 重组酶就会介导靶基因两侧的1oxP 间发生切除反应，结果将一个loxP 和靶基因切除。这样，靶基因的修饰(切除)是以Cre 的表达为前提的。Cre 的表达特性决定了靶基因的修饰(切除)持性：即Cre 在哪一种组织细胞中表达，靶基因的修饰(切除)就发生在哪种组织细胞；而Cre 的表达水平将影响靶基因在此种组织细胞中进行修饰的效率。所以只要控制Cre 的表达特异性和表达水平就可实现对小鼠中靶基因修饰的特异性和程度。

       One problem with a simple, constitutive targeting strategy is that many genes have multiple functions, or are active in multiple tissues and/or at multiple stages of development. Many knockouts of genes with no known roles in development have resulted in embryonic lethality, preventing the study of the gene’s role in the phenotype of an adult animal. To get around this problem, techniques have been developed to allow the investigator to determine when and where the knockout occurs. Conditional targeting constructs employ recombinase recognition sequences, tissue-specific promoters, developmentally-specific promoters, or inducible promoters (or a combination of these) to limit and control the spatial and temporal expression of the knockout or knock-in phenotype.

    I. Excision reaction:

When the loxP sites are oriented in the same direction, Cre brings the two loxP sites together and removes the intervening DNA along with one loxP site, in the form of a circular molecule.  The circular DNA product is then degraded by the cell, so this reaction is essentially irreversible.  However, driving the reaction in reverse is possible and allows the insertion of circular DNA molecules containing a loxP site into a locus containing a single loxP site.

The product of the excision reaction above is an allele lacking exon 2, which could produce a partially active protein product.  However, if splicing between exons 1 and 3 results in a frameshift, the resulting mRNA is likely to be destroyed by the nonsense-mediated decay (NMD) pathway, resulting in a null phenotype.

 

II.  Inversion reaction:

When the loxP sites are oriented in opposite directions, Cre will invert the intervening sequence and leave both loxP sites intact.  With wildtype loxP sites, this reaction is reversible.  To make the reaction irreversible, mutant loxP sites have been developed such that, after Cre acts on them, they will be inactive.  This strategy is made possible by the fact that Cre's mechanism involves pairing the loxP sites with each other, cleaving them within the 8bp core sequence, and causing a crossover at the cleavage site.  When mutant loxP sites are used, the crossover produces an 8bp core that is no longer recognized by Cre.

Most modern targeting constructs, including many of those used by members of the International Knockout Mouse Consortium (KOMP, EUCOMM, NorCOMM, etc.), use both Cre and Flp recognition sites.  One common motif of such constructs is illustrated in the following diagram, where the neomycin resistance cassette is flanked by frt sites while exon 2 is flanked by loxP sites.

The reason for removing the neomycin resistance cassette is that, even though it resides in an intron, it can have unpredictable effects on the expression of the gene of interest and other nearby genes. For example, it may stimulate inappropriate mRNA splicing events, or its promoter may affect downstream genes.  (These effects can also be mitigated by inserting the neo cassette in a reverse orientation to the gene of interest.)

Normally, chimeric mice are made by injecting blastocysts with ES cells that have been targeted with the first allele shown above (containing 2 frt sites and 2 loxP sites).  If the chimeras are crossed with Flp-expressing transgenic mice, some of their offspring will have the second allele pictured above (one frt site and 2 loxP sites).  These animals are said to have a floxed gene, meaning the gene of interest has one or more exons flanked by loxP sites (flox = flanked by loxP).  (The Flp reaction can also be accomplished in vitro, by electroporating a Flp-expressing plasmid into the ES cells.  However, we do not recommend this strategy because it subjects the ES cells to additional time in culture that may compromise their ability to contribute to the germline of chimeras.)

Except for the presence of the frt site and 2 loxP sites, the floxed mice have an otherwise completely wildtype allele that can be knocked out by the expression of Cre inside their cells (assuming removal of the second exon results in a knockout). Breeding a floxed mouse with a mouse that expresses Cre from a transgene will produce some offspring that inherit both the floxed allele and the transgene. These mice will lack the second exon of the gene of interest on one chromosome, due to the action of Cre on the two loxP sites, as illustrated in the third allele above. Further crosses between floxed mice and flox/Cre mice will result in a homozygous knockout.

The real power of this system becomes apparent when one considers the fact that multiple lines of Cre-expressing mice are available, with different promoters driving expression of Cre. If the promoter is neuron-specific, for example, the knockout will occur only in neurons. Cre expression itself can be made conditional (e.g., by using a tetracycline-responsive promoter), allowing the exact time at which the gene is knocked out to be determined by the investigator.

   注：主要来源于http://www.research./tmf/index.htm

   1、Cre／loxP系统

原理：Cre／loxP系统来源于F1噬菌体，可以介导位点特异的DNA重组。该系统含有两种成分：①一段长34bp的DNA序列，含有两个13 bp的反向重复序列和一个8 bp的核心序列。这段34bp序列是重组酶识别的位点，被称为loxP位点(10cus of X—over in P1)。②Cre重组酶(cyclizationrecombination)，它是一种由343个氨基酸组成的单体蛋白，可以引发loxP位点的DNA重组。

    优点：Cre／10xP系统之所以在基因敲除中获得了非常广泛的应用，是由该系统的诸多优点决定的：①Cre重组酶与具有loxP位点的DNA片断形成复合物后，可以提供足够的能量引发之后的DNA重组过程，因此该系统不需要细胞或者生物体提供其他的辅助因子；②loxP位点是一段较短的DNA序列，因此非常容易合成；③Cre重组酶是一种比较稳定的蛋白质，因此可以在生物体不同的组织、不同的生理条件下发挥作用；④Cre重组酶的编码基因可以置于任何一种启动子的调控之下，从而使这种重组酶在生物体不同的细胞、组织、器官，以及不同的发育阶段或不同的生理条件下产生，进而发挥作用，这一点也是该系统在应用过程中最为重要的一点。

    工作流程：利用Cre／loxP系统实现体内某特定基因在特定条件下的敲除，需要两只转基因小鼠。第一只小鼠一般采用胚胎干细胞技术获得，首先在体外构建一个在目的基因两端分别含有一个loxP位点的基因序列，之后将体外构建好的这段基因序列转入胚胎干细胞内，使其通过同源重组替代细胞基因组内原来的基因序列。经过这样处理的胚胎干细胞被重新植入到假孕小鼠的子宫内，使其重新发育成为一个完整的胚胎，最终成为一只转基因小鼠。在这只转基因小鼠中，loxP位点被引入到相应基因的内含子内，理论上不会对相应基因的功能产生影响，因此一般情况下，该小鼠的表型是正常的。第二只转基因小鼠一般采用卵母细胞注射或者胚胎干细胞技术获得，在这只小鼠中，Cre重组酶被置于某特定基因启动子的调控之下，可以使其在某特定的条件下表达。最后，让这两只小鼠进行交配，产生的同时含有上述两种基因型的子代小鼠就会在某一特定类型的细胞中缺失某一特定的基因。

    很明显，在何种组织细胞或器官中敲除某一特定的基因取决于所选择的启动子。只要选择合适的启动子调控Cre重组酶的表达，使其在生物体特定的部位、特定的条件下产生，就可以实现相应条件下某一特定基因的敲除。迄今为止，研究者们已经成功地利用多个不同的启动子实现了在不同条件下的基因敲除，这些启动子可以是细胞类型特异的，如lck启动子(胸腺细胞)、alphaA晶状体球蛋白启动子(眼晶状体)、钙调素依赖性激酶Ⅱ启动子(海马和大脑新皮质)、乳清酸性蛋白启动子(乳腺)、aP2启动子(脂肪组织)、AQP2启动子(肾脏集合管)和肌浆蛋白启动子(骨骼肌)等。启动子也可以受某些外源性化学物质的调控，外源性调控的基因敲除可以避免在胚胎发育早期由于基因功能的异常所产生的副作用，如干扰素反应Mxl启动子、他莫西酚依赖的雌激素突变体启动子和四环素调节系统等。

    理论上，Cre／loxP系统介导的条件性基因敲除具有对任何发育阶段的任何组织细胞中的任何基因进行功能研究的潜力。

    2、Flp／FRT系统

    该系统与Cre／loxP系统相同，也是由一个重组酶和一段特殊的DNA序列组成。从进化的角度上考虑，Flp／FRT系统是Cre／loxP系统在真核细胞内的同源系统。其中．重组酶Flp是酵母细胞内的一个由423个氨基酸组成的单体蛋白。与Cre相似，Flp发挥作用也不需要任何辅助因子，同时在不同的条件下具有良好的稳定性。该系统的另一个成分Flp识别位点(Flp recognition target，FRT)与loxP位点非常相似，同样由两个长度为13bp的反向重复序列和一个长度为8 bp的核心序列构成。在该系统发挥作用时，FRT位点的方向决定了目的片段的缺失还是倒转。这两个系统比较明显的区别是它们发挥作用的最佳温度不同，Cre重组酶发挥作用的最佳温度为37℃，而Flp重组酶为30℃。因此，Cre／loxP系统最适宜在动物体内使用。loxP和FRT位点的序列如图所示。

注：主要来源于：http://www./web/page/news6854.htm