Functional Genomics & RNAi

TargeTron® Resources

Detailed Explanation of the TargeTron System

The TargeTron Gene Knockout System provides optimized reagents and protocols for the rapid and specific disruption of bacterial genes by insertion of group II introns. Unlike conventional DNA transposon mutagenesis, the TargeTron method is site-specific, not random. Recent advances in group II intron research have enabled re-targeting of introns to insert efficiently into virtually any desired DNA target 1,2,3,4. Features of the TargeTron retrohoming mechanism make it attractive for genetic manipulation. First, retrohoming is highly efficient and specific. Retrohoming frequencies can approach 100% and are six orders of magnitude higher than the frequency of ectopic integration. Second, the introns are minimally dependent on host factors, making them applicable to a broad range of bacteria. To date, the TargeTron system has been validated in Escherichia coli 1, Staphylococcus aureus, Clostridium perfringens, Shigella flexneri 3, Salmonella typhimerium 3, and Lactococcus lactis 2.

Escherichia coli Gene Knockouts Created with the TargeTron System Validated by DNA Sequencing

bcr malM recQ yfjK
carB mfd rep ygcB
dadA nagA rhlB ygcB
dbpA ndk rhlE yhjU
deaD nuoE me yoaA
deoR phoH srmB  
dinG phoH thyA  
endA priA trpE  
fadR proA uvrB  
helD rbfa uvrD  
hepA recB ybeZ  
hrpA recC ycac  
hrpB recD yecE  
lhr recG yejH  

Group II introns insert themselves via the activity of an RNA-protein complex (RNP) expressed from a single plasmid provided in the kit. The RNA portion of the RNP is easily mutated via PCR to re-target insertion into a user-specified chromosomal gene. A somewhat accurate analogy is that group II introns are like programmable restriction enzymes, with the added activity of inserting RNA into a cleaved DNA sequence.

The diagram overview of method outlines the process of using the TargeTron kit to knockout bacterial genes. First, a computer algorithm (http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/targetron.html) is used to identify target sites in your gene of interest. A password provided in the kit will allow the user to enter individual gene sequences into the algorithm for output of recommended primer sets as ranked design options. To pre-screen your target sequence prior to purchase, please contact Technical Service. A typical 1 kb gene can be expected to contain 5 to 11 group II intron insertion sites (design options). One or more of the primer set options may be used to mutate (re-target) the intron RNA component (provided in the kit) by PCR. Next, the mutated 350 bp PCR fragment is ligated into a linearized vector that contains the remaining intron components. The ligation reaction is transformed into the host, and the re-targeted intron expressed.

The resulting RNP complex scans the host genomic DNA for its specific insertion site. The intron RNA inserts into the gene of interest followed by reverse transcription and repair to create the permanent double-strand DNA disrupted mutant. Knockouts are then selected using a kanamycin marker that is activated upon chromosomal insertion. Using gene specific primers, kanamycin resistant colonies are PCR screened to confirm insertion.

View the Technical Manual (1.16 Mb PDF)

View the pACD4K-C Vector Sequence

 

Diagram Overview of Method


 

TargeTron Algorithm

Each TargeTron kit provides an access card and password for a specified number of “designs” (3EA or 10EA). The password gives the user access to the extensively validated TargeTron algorithm that predicts optimal intron insertion sites and designs primers for mutating the intron to insert into those sites. The algorithm is extremely simple to use, requiring only the input of the target gene to be knocked out (a design). Various options for knocking out the gene sequence are instantly returned to the user in the form of suggested oligo sequences for mutating the group II intron sequence. The options are specific points of insertion and are ranked in order of expected highest efficiency. After selection of one or more desireable insertion points, the required oligos are ordered to begin laboratory procedures using the kit. Contact techserv@sial.com to pre-screen genes for potential insertion sites before purchasing a kit or using a password.

 

References

  1. Perutka, J., Wang, W., Goerlitz, D., Lambowitz, A.M. 2004. J. Mol. Biol. 336(2):421-39.
  2. Frazier, C.L., San Filippo, J., Lambowitz, A.M., Mills, D.A. 2003. Appl. Environ. Microbiol. 69(2):1121-8.
  3. Karberg, M., Guo, H., Zhong, J., Coon, R., Perutka, J., Lambowitz, A.M. 2001. Nat. Biotechnol. 19(12):1162-7.
  4. Zhong, J., Karberg, M., Lambowitz, A.M. 2003. Nucleic Acids Res. 31(6):1656-64.

This product and its use are the subject of one or more of U.S. Patent Nos. 5,698,421, 5,804,418, 5,869,634, 6,027,895, 6,001,608, and 6,306,596 and/or other pending U.S. and foreign patent applications controlled by InGex, LLC.
TargeTron is a registered trademark of InGex, LLC.

back to TargeTron Gene Knockout System