A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes
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Since their first description in 1991 (1 ), CAG-disease causing genes are increasing in number. Up to date, there are at least nine genetic diseases caused by CAG expansions. The creation of transgene and knock-in mice with CAG expansions is an useful tool for understanding the pathological mechanisms of the corresponding diseases, which could lead to therapeutic target(s) for the diseases. However, owing to the short life expectancy of the mice and to the low expression levels of transgenes, it is necessary to introduce CAG expansions larger than that in humans in order to elicit a pathological phenotype within the life of mice models. Naturally occurring “huge” CAG expansions (>100-150 CAGs), which could induce disease phenotype in the mice, are very seldom. In vitro synthesis of isolated CAG repeats have already been described (2 ,3 ). Most of these methods, however, require further cloning steps and often contain some flanking extraneous sequences. Here, the author describes a fast and simple way for expanding/introducing CAG repeats (or other repeats!) without altering the flanking 5′ and 3′ sequences of the gene of interest. This method was successfully employed for expanding the CAG repeat of the MJD/SCA3 gene (4 ). Fig. 1 outlined the strategy of this method. Two independent polymerase chain reactions (PCRs) amplify the target gene from 5′ to the CAG repeat region (PCR I) and from the CAG repeat to the 3′ region (PCR II) of the gene. The amplicons of PCR I and PCR II will be then mixed, elongated and then a third PCR will be carried out with the two most “outsider” primers. We used this strategy for elongating the CAG of the MJD/SCA3 gene from 22 up to 138 CAG repeats (see Figs. 2 and3 ).
Fig. 1. Strategy for introducing CAG expansions into CAG-containing genes. (A) Diagrammatic representation of the target sequence including the repeat region (CAG repeat), restriction sites X and Y, where X is the enzyme for the “5′-digestion” and Y is the enzyme for the “3′-digestion” as explained in the text and the two outsider specific forward primer (FOR-1) and reverse primer (REV-1). (B) amplification of the target DNA in two distinct reactions after digestion with the X and Y restriction endonucleases in case of a circular template. As primers the pair FOR-1/(CTG)7 for PCR I and the pair (CTG)7/REV-1 for PCR II were used. (B2, B3, B4), subsequent cycles of PCR I leading to a progressive elongation of the repeat. (C) Amplicons of both PCRs. (D) Aliquots of both PCRs were mixed, denatured, annealed and extended with DNA polymerase. (E) Amplification of the elongated products of step D in PCR III with the specific (vector or gene specific) primers FOR-1 and REV-1. Arrowhead: variable CAG repeats. (Slightly modified from Laccone et al. (4 ), reprinted by permission of Wiley-Liss, Inc. Jossey-Bass Inc., a subsidiary of Wiley and Sons, Inc.)
Fig. 2. (A) Electrophoresis of the PCR products (7 μL each) on a 1% agarose gel of the MJD/SCA3 cDNA. Lane-1: 1-kb-ladder; Lane 2: amplification product of the complete 1.4-kb cDNA (CA, control amplification) obtained with the primers (FOR-1 and REV-1); Lane-3: PCR I product of about 1.1 kb (primers FOR-1/(CAG)7); Lane 4: PCR II product obtained with primers primers (FOR-1 and REV-1); Lane-3: PCR I product of about 1.1 kb (primers FOR-1/ (CAG)7); Lane 4: PCR II product obtained with primers (CTG)7/REV-1. The visible smear in both amplicons of PCR I and II should represent most probably a continuous expansion of the repeat region. (B) Lane 1: 1-kb ladder; lane 2: amplification of the target cDNA with the FOR-1 and REV-1 specific primers. Lane 3-5: results of PCR III obtained by mixing 1, 2, and 4 μL of PCR I and PCR II amplicons with the two specific primers FOR-1 and REV-1. The visible smear represents most probably a series of products with elongated CAGs. In lane 2 the faint band of about 4.4 kb (AA = additional amplicons) may be caused by the linear amplification of the target circular clones which in this case have been not digested prior to the PCR. (From Laccone et al. (4 ), reprinted by permission of Wiley-Liss Inc. Jossey-Bass Inc., a subsidiary of Wiley and Sons, Inc.)
Fig. 3. (A ) Eco RI/Hind III digestion of the cloned products of PCR III to release the plasmid insert. Because the MJD/SCA3 gene contains an Eco RI restriction site at position 435, we expected a constant fragment of about 450 bp (arrowhead) and a fragment of 700+(CAG)n bp (arrow) attributable to the presence of CAGs variable in size. (B ) Hybridzation of the blotted plasmid DNA with a probe containing 63 CAGs. (from Laccone et al. (4 ), reprinted by permission of Wiley-Liss Inc. Jossey-Bass Inc., a subsidiary of Wiley and Sons, Inc.).
