Gene Targeting Guide

This Gene Targeting Guide for knock-out first allele constructs is similar to the following strategies used by different consortia:

© knockoutmouse.org

For a strategy overview of our Gene Targeting pipeline, please see section 15)

Look up your gene of interest
in ENSEMBL (and UCSC)

Example: Ctr9 ( ENSMUSG00000005609)

• note down gene names, accesion number and other gene identifiers (location, protein features)
• look up whether there is already a Knock--Out Cell Line or Targeting Vector available at International Mouse Phenotyping Consortium (IMPC)

Is the gene of interest expressed in ES-cells?

• check with the International Gene Trap Consortium (IGTC) for existing cell lines
• expressed genesd are suitable for promoterless targeting cassettes

Example: Ctr9 has 2 different gene traps (pic)

Selection of a target exon for a knock-out construct

• view exons of the transcript you have chosen as design basis (pic)
• look for the most 5' exon that meets the following criteria

* 500 bp is the minimum size of flanking introns in the EUCOMM designs, 150 bp is usually enough to put a cassette without interruption of splice adaptors and splice donors (leave at least 50 bp from the splice donor, which is 5' end of the intron, and 100 bp from the splice acceptor, which is the 3' end of the intron)

**those criteria are taken in account by EUCOMM designers and are not the most important for us

• note down exon identifier and position

Exceptions:

• If flanking introns are too small or the exon itself is very small, try to knock out 2 sequential exons (check for frame shift again)
• For one intron genes, put the downstream loxP-site into the 3' non-coding region or downstream of the polyadenylation signal

Examination of the critical exon's environment

• view chosen exon and at least 5 kb up and downstream in Location view (pic)
• check for regulatory elements and repeats in this region

• to define the positions of the cassettes as well as the homology arm length (by subcloning), you have to select oligos of 50 bp [70 bp]* which serve as homology arms in recombineering
• to design those oligos you can use the
• recheck also the G-oligos for not recombining in exons (if small deletions happen) and in CpG-island (chromosome structure might inhibit recombination)

HTGT-tool

• create an account under http://www.sanger.ac.uk/htgt 
• use custom design tool
• create a design for knock out first, block specified (means not fixed to specific bp)
• set parameters (taking intron sizes, repeats, HCNEs, ... in account)

changes of default for Ctr9 (pic)
     homology arm length: 5000 bp
     upstream spacer: 250 bp 

• insert Ensembl ID: ENSMUSG00000005609
• select critical exon(s):  ENSMUSE00000203310
• change comment line (otherwise your email address is visible with the designs in ENSEMBL)
• Create&Run
• note down ID 
• click refresh or check for design by ID later (pic)
• in case design failed or you want to change it, click Edit and change parameters as described above (you may delete the old design then)

Manual Design

• neccessary for non-mouse DNA, non-exon targeting or non-annotated genes
• download gene sequence into your vector construction program
• select the positions in the same distance from the critical exon as for the HTGT-tool
• blast about 150 bp to the genome
• find non-repetetive regions (exclude low complexity sequence)
• check for around 50% GC content
• (exclude hairpin formation etc. if software available)
• decide on 50 bp* serving as oligo (homology arm for recombineering)

*70 bp should be used for subcloning oligos.

Retrieving the sequence

• click on critical exon to get it in the Ensembl location view
• export data, add 20,000 bp flanking sequence on either side (necc. for southern design)
• export as genbank file if you vector program has an import function for that
• otherwise export as text and manually annotate
• in case you used the HTGT tool you can also copy and paste the 15_E_15 sequence, which is exon plus 15 kb up and downstream sequence

© Madeleine Walker

Annotation of the oligos in your file

• mark the oligos in the genomic sequence
• see where homology arms will end and which small intronic regions will be deleted by the cassette insertion (keep in mind for southern strategy)

© Madeleine Walker

Creation of the targeted allele

• create a new file and place your cassettes
• 1st cassette here: FRT-SA-GT1-T2A-lacZneo-CotC-FRT-loxP 
• 2nd cassette here: rox-PGK-HYG-rox-loxP
• ATTENTION: always check the orientation of the loxP-sites as different cassettes bring loxP-sites in different orientations. Only loxP sites that point in the same direction will give the intended deletions.

© Madeleine Walker

• early southern strategy design is recommended (certain designs may have no good possibilities for southern probes or enzymes and therefore have to be altered, best before practical work starts)
• design the southern strategy by comparing wildtype allele to targeted allele

Finding southern probes

• blast about 1000 bp left of 5' homology end and right of 3' homology end to the mouse genome (set blast to "no filter")
• find at least 500 bp continuous sequence without repeats and create primers in it, that give a PCR product of 500-700 bp (good southern probe size)
• the closer the probe is next to the ends of the homology, the bigger is the chance to find good enzymes
• probes that stretch over exons are favourable

© screenshot

Finding restriction enzymes

that cut neither in the homology arms nor in the southern probes, meaning for the 5' southern you pick enzymes that do not cut in the region between the 5' fwd primer of the probe and oligo U5, and for the 3' southern enzymes that do not cut between oligo D3 and 3' rev primer of the probe (pic) 
               that are not inhibited by eucaryotic methylation patterns (table)
               that give a band (when hybridized with the labelled probe) of 5 to 15 kb
               that give a size difference between WT and targeted allele of > 1kb

• here: for 5' ScaI gives a band at 9888 bp for WT and 11881 bp for the targeted allele
• have at least one alternative in case enzyme cuts unexpectedly
• you may do double digests if no simple digest meets the criteria (check for enzyme buffer compatibility)
• create a theoretical gel picture for the outcome of the southern showing the size of the signal after developing the membrane (pic)
• repeat all steps for the 3' southern

If you cannot find nice probe regions or good enzymes, try to redesign the homology arms.

Creating the targeting vector in silico

• copy the sequence between the homology arm ends from the targeted allele and paste it to the backbone 

© madeleine Walker

Linearisation Sites

• check for restriction sites in the backbone for linearisation
• use (combination of) restrictions sites that cut out backbone (example: SgrAI)
• or insert a new restriction site into junction between homology arm and backbone on both sides (for using the counter selection marker on the backbone [TK/DTA] only on one side)
• example for Ctr9: SalI (GTCGAC) or SnaBI (TACGTA)
• note down on which side the counter selection marker will remain after digestions

© Madeleine Walker

Oligos for ordering

• get the complete sequence of the oligo by copying the 50/70 bp homology (designed by HTGT) plus the primer for cassette/backbone amplification
• [please note that for subcloning by gap repair (see C in figure below), the sequences included in the oligonucleotides should be the inverse complement to the orientations which are for insertions (see B in figure below)]
• by copying it out of the final file, there is no chance of miscombining the 2 parts
• do not forget to make the reverse complement of the downstream primer

© Madeleine Walker

Important information
→ Overview of mouse and human BAC libraries including informations about DNA origin, backbone and selection markers. 

Finding a BAC and ordering it

• we recommend to use UCSC to find BACs, as they only show end-sequenced BACs and have prooven by experience to be more reliable in the annotation (we rarely see BACs with the same name, having different length in the 2 browsers, especially for human)
• to display the BACs:

Example for Ctr9: RP23-463L18 (118,072,655-118,240,067)

There is also a clone finder at NCBI, that reads out many more libraries. We have no experience with its completeness and reliabily though so far.

Features for a good BAC

• BAC needs to contain at least the critical exon plus 5 kb up and downstream
• better: find a BAC that contains the whole gene plus 5'UTR, so that it permits usage for BAC transgenesis projects as well as for targeting issues
• the BAC should not be too big, better go for 150 kb than 300 kb, because internal rearrangements are less likely
• search for the BAC in NCBI and take one that is at least sequence confirmed on one side (SP6 or T7), better on both (use the UCSC browser to find only BACs with end-sequencing information, use the direct link from the BAC to NCBI to look up the end-sequencing information)

Creating the BAC map

• when you have found the best BAC, right click on it and get its end points
• export the sequence between this endpoints (for Ensembl: location view -> export data -> no flanking sequence)
• this sequence is the insert sequence
• if you want information about the BAC library backbones, see table or visit CHORI website
• put the sequence into your DNA depiction software and annotate

© Madeleine Walker

• create all intermediate files from the WT BAC file to final subclone according to the actual strategy in the lab
• use these files to decide on enzymes for restriction analysis for the intermediate steps (detection of deletions or mixtures!)

For ssOligo-experiments and backbone modifications (e.g. PiggyBac integration)
-> Overview of backbone maps and replication orientation.

What needs to be genotyped in the modified ES-cells/mouse?

• checking for the presence of the 3' loxP (especially if it is a single loxP without selection marker)
• checking for successfully Flped allele (loss of lacZneo-cassette)
• checking for successfully knocked out allele (loss of critical exon)

Where to design primers?

• put primers into WT sequence and distinguish the WT from the targeted by size difference - in case of a heterozygous gene (+/-, +/F, ...) you will always get both bands
• in case of low size difference or huge distance of primers, put an additional primer into the cassette/loxP and prove the existence of the cassette/loxP by getting a product

make the primer longer than 21 nt and have 3 non-matching nt at the end with a C/G at the very 3' end

Detailed description of the most important cassettes:

FRT-SA-IRES-lacZneo-pA-FRT-loxP (Chen and Soriano, 2003)

This stop cassette includes the Engrailed-2 splice acceptor to capture the transcript of the target gene. It is followed by an internal ribosome entry site (IRES) promoting the expression of a b-galactosidase-neomycin resistance fusion protein and a 3'-non-coding region ending in a polyadenylation signal. The neomycin resistance gene has an additional bacterial promotor (in frame). The cassette is flanked by FRT sites, allowing to restore gene function after the trapping by its removal via Flp expression. The loxP-site is unaffected by this recombination event.

FRT-SA-GT0/1/2-T2A-lacZneo-pA-CoTC-FRT-loxP

This cassette is the new version of the previous trapping cassette. The splice acceptor has been shortened and the IRES has been replaced by a 2A ribosomal-error peptide (Szymczak et al, 2004). This makes the cassette smalle but the absence of an IRES requires the cassette to be in frame with the exon upstream of it. Consequently the stop cassette has been made in three versions. During design, the correct version needs to be selected according to the 0,1 or 2 reading frame of the upstream exon. A second 2A peptide is included 3' of b-galactosidase, so that it is a separate protein from neomycin resistance, which enhances performance of both proteins compared to the fused version. In addition to the poly adenylation signal the cassette has a co-transcriptional cleavage site. The position of the FRT and loxP sites is as described. 

rox-BSD-Em7-PGK-rox-loxP

 This cassette introduces the 3?loxP site into the 3?intron. It includes a PGK promoter driving the eucaryotic and an Em7 promotor driving the bacterial expression of the blasticidin resistance gene. For genes that are expressed in ES cells and can be targeted by selection of neomycin/G418 resistance driven from the endogenous target gene promoter (termed ?promoterless selection?), the rox cassette should be removed in E.coli by expressing Dre recombinase from pSC101-BAD-Dre (Anastassiadis et al, 2009) to leave the 3?loxP site in the targeting construct. However, in a few cases after targeting in ES cells, we have found that all targeted clones omit the 3?loxP site. In these cases, the targeting can be repeated using the construct including the rox cassette and selecting for both neomycin and blasticidin resistance to force the inclusion of the 3?loxP site. For genes that are not expressed in ES cells, the targeting construct must include a promoter to drive expression of the selectable gene. Here the rox cassette provides this function. In the cases when the rox cassette remains after targeting, it must be removed either by expressing Dre in ES cells or by crossing to a Dre deleter (Anastassiadis et al, 2009).

p15A-HSVtk-DTA-amp

The subcloning vector, p15A-HSKtk-DTA-ampR, is based on the p15A plasmid origin. Consequently it does not give the very high copy yields delivered by the mutated colE1 origins in pUC-type plasmids. We have found that these unnaturally very high copy plasmids can provoke recombineering problems, so prefer p15A or the unmutated colE1 origin from pBR322 for recombineering applications, particularly subcloning by gap repair. In p15A-pTK-DTA-ampR, the diptheria toxin A chain (DTA) coding region under the HSVtk promoter lies between the p15A origin and the ampicillin resistance gene. For subcloning, the vector is amplified by PCR to attach the homology arms. The presence of a DTA gene permits the use of positive/negative selection (Mansour et al, 1988; Yagi et al, 1990). We have found that negative selection can benefit promoter-driven selection but has little effect on promoterless selection. Therefore we recommend that the vector be entirely cut off for promoterless selection or linearized for promoter-driven selection.

Gene Targeting Pipeline for Standard Conditional Alleles

© Madeleine Walker

PCReactions and Cassette Preparation

Three PCR products are required to generate the first allele targeting construct. 

The first PCR product is amplified from the rpsL-gentamycin selectable/counterselectable cassette. The PCR amplified rpsL-gene cassette already contains the homology arms for the stop cassette (step 3). Consequently incorporation of the rpsL-gene cassette into the BAC by selection for gentamycin resistance incorporates the homology arms for the stop cassette. We include this step because it avoids PCR amplification of the 6.1kb stop cassette. Instead a restriction fragment from a pR6K-amp-lacZneo plasmid preparation is used, thereby reducing unwanted, PCR-based, mutagenesis. 

The second PCR product is the amplification of the subcloning vector, p15A-pTK-DTA-amp^R. 

The third PCR product is amplified from the pR6K-PGK-BSD template, which will insert a loxP site on the 3' side of the chosen exon.

*it is possible to shorten these oligos (however, do not shorten the amplified homology arms for the stop cassette)

For further instructions concerning PCR conditions and the subsequent recombineering pipeline, please see our book chapters in Methods in Enzymology. 

For plasmid requests, see here. 

The following publications have been cited in our Enzymology Book Chapter about the Gene Targeting Pipeline. For further publications related to Gene Targeting, use of Site Specific Recombinases or Recombineering, please see our publications.

Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F. and Stewart, A. F. (2009). "Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice." Dis Model Mech 2: 508-15.

Branda, C. S. and Dymecki, S. M. (2004). "Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice." Dev Cell 6: 7-28.

Buchholz, F., Angrand, P. O. and Stewart, A. F. (1996). "A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs." Nucleic Acids Res 24: 3118-9.

Chen, W. V. and Soriano, P. (2003). "Gene trap mutagenesis in embryonic stem cells." Methods Enzymol 365: 367-86.

Copeland, N. G., Jenkins, N. A. and Court, D. L. (2001). "Recombineering: a powerful new tool for mouse functional genomics." Nat Rev Genet 2: 769-79.

Court, D. L., Sawitzke, J. A. and Thomason, L. C. (2002). "Genetic engineering using homologous recombination." Annu Rev Genet 36: 361-88.

Datsenko, K. A. and Wanner, B. L. (2000). "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products." Proc Natl Acad Sci U S A 97: 6640-5.

Ellis, H. M., Yu, D., DiTizio, T. and Court, D. L. (2001). "High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides." Proc Natl Acad Sci U S A 98: 6742-6.

Filutowicz, M., McEachern, M. J. and Helinski, D. R. (1986). "Positive and negative roles of an initiator protein at an origin of replication." Proc Natl Acad Sci U S A 83: 9645-9.

Glaser, S., Anastassiadis, K. and Stewart, A. F. (2005). "Current issues in mouse genome engineering." Nat Genet 37: 1187-93.

Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P. and Kushner, S. R. (1989). "New method for generating deletions and gene replacements in Escherichia coli." J Bacteriol 171: 4617-22.

Hashimoto, T. and Sekiguchi, M. (1976). "Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine." J Bacteriol 127: 1561-3.

Ivics, Z., Li, M. A., Mates, L., Boeke, J. D., Nagy, A., Bradley, A. and Izsvak, Z. (2009). "Transposon-mediated genome manipulation in vertebrates." Nat Methods 6: 415-22.

Mansour, S. L., Thomas, K. R. and Capecchi, M. R. (1988). "Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes." Nature 336: 348-52. 

Poser, I., Sarov, M., Hutchins, J. R., Heriche, J. K., Toyoda, Y., Pozniakovsky, A., Weigl, D., Nitzsche, A., Hegemann, B., Bird, A. W., Pelletier, L., Kittler, R., Hua, S., Naumann, R., Augsburg, M., Sykora, M. M., Hofemeister, H., Zhang, Y., Nasmyth, K., White, K. P., Dietzel, S., Mechtler, K., Durbin, R., Stewart, A. F., Peters, J. M., Buchholz, F. and Hyman, A. A. (2008). "BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals." Nat Methods 5: 409-15.

Ringrose, L., Chabanis, S., Angrand, P. O., Woodroofe, C. and Stewart, A. F. (1999). "Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances." EMBO J 18: 6630-41.

Sarov, M., Schneider, S., Pozniakovski, A., Roguev, A., Ernst, S., Zhang, Y., Hyman, A. A. and Stewart, A. F. (2006). "A recombineering pipeline for functional genomics applied to Caenorhabditis elegans." Nat Methods 3: 839-44.

Sauer, B. and McDermott, J. (2004). "DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages." Nucleic Acids Res 32: 6086-95.

Sawitzke, J. A., Thomason, L. C., Costantino, N., Bubunenko, M., Datta, S. and Court, D. L. (2007). "Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond." Methods Enzymol 421: 171-99.

Skarnes, W., Rosen, B., West, A., Koutsourakis, M., Bushell, W., Iyer, V., Cox, T., Jackson, D., Severin, J., Biggs, P., Thomas, M., Mujica, A., Harrow, J., Fu, J., Nefedov, M., de Jong, P., Stewart, A. and Bradley, A. (2010). "A conditional knockout resource for genome-wide analysis of mouse gene function." Nature in press.

Szymczak, A. L., Workman, C. J., Wang, Y., Vignali, K. M., Dilioglou, S., Vanin, E. F. and Vignali, D. A. (2004). "Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector." Nat Biotechnol 22: 589-94.

Testa, G., Schaft, J., van der Hoeven, F., Glaser, S., Anastassiadis, K., Zhang, Y., Hermann, T., Stremmel, W. and Stewart, A. F. (2004). "A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles." Genesis 38: 151-8.

Testa, G., Zhang, Y., Vintersten, K., Benes, V., Pijnappel, W. W., Chambers, I., Smith, A. J., Smith, A. G. and Stewart, A. F. (2003). "Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles." Nat Biotechnol 21: 443-7.

Valenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, N. W., Economides, A. N., Auerbach, W., Poueymirou, W. T., Adams, N. C., Rojas, J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser, A. L., Esau, L., Zheng, J., Griffiths, J. A., Wang, X., Su, H., Xue, Y., Dominguez, M. G., Noguera, I., Torres, R., Macdonald, L. E., Stewart, A. F., DeChiara, T. M. and Yancopoulos, G. D. (2003). "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis." Nat Biotechnol 21: 652-9.

Wang, J., Sarov, M., Rientjes, J., Fu, J., Hollak, H., Kranz, H., Xie, W., Stewart, A. F. and Zhang, Y. (2006). "An improved recombineering approach by adding RecA to lambda Red recombination." Mol Biotechnol 32: 43-53.

Wu, S., Wu, Y. and Capecchi, M. R. (2006). "Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo." Development 133: 581-90.

Wu, S., Ying, G., Wu, Q. and Capecchi, M. R. (2008). "A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond." Nat Protoc 3: 1056-76.

Yagi, T., Ikawa, Y., Yoshida, K., Shigetani, Y., Takeda, N., Mabuchi, I., Yamamoto, T. and Aizawa, S. (1990). "Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection." Proc Natl Acad Sci U S A 87: 9918-22.

Yang, X. W., Model, P. and Heintz, N. (1997). "Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome." Nat Biotechnol 15: 859-65.

Yang, Y. and Seed, B. (2003). "Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes." Nat Biotechnol 21: 447-51.

Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G. and Court, D. L. (2000). "An efficient recombination system for chromosome engineering in Escherichia coli." Proc Natl Acad Sci U S A 97: 5978-83.

Zhang, Y., Buchholz, F., Muyrers, J. P. and Stewart, A. F. (1998). "A new logic for DNA engineering using recombination in Escherichia coli." Nat Genet 20: 123-8.

Zhang, Y., Muyrers, J. P., Testa, G. and Stewart, A. F. (2000). "DNA cloning by homologous recombination in Escherichia coli." Nat Biotechnol 18: 1314-7.

AMP        Ampicillin
 AOS        Array Oligo Selector
 ATG         Codon for the initiating methionine
 ATT         Gateway attachement site (attB, attP, attR, attL)
 BAC         Bacterial Artificial Chromosome
 bb         Backbone of a plasmid (origin of replication + selectable marker)
 BLAT         BLAST Like Alignment Tool
 BLAST         Basic Local Alignment Search Tool
 BSD         Blasticidin
 CCDS         Consensus CDS
 CDS         Coding sequence for rotein
 CE         Critical Exon
 CHORI         Children?s Hospital Oakland Research Institute
 CM         Chloramphenicol
 CNV         Copy Number Variant
 CoTC         Co-Translational Cleavage signal
 Cre         Site-specific recombinase from bacteriophage P1 genome
 CSD         CHORI, Sanger Institute, and UC Davis
 csm         Counter selection marker
 D         Downstream (oligo)
 DAS         Distributed Annotation System
 Dre         Phage D6 derived site-specific DNA recombinase
 EGFP         Enhanced Green Fluorescent Protein
 EJC         Exon Junction Complex
 EBI         European Bioinformatics Institute
 EMBL         European Molecular Biology Laboratory
 EMMA         European Mouse Mutant Archive
 ENCODE         Encyclopedia of DNA Elements
 EST         Expressed Sequence Tag
 EuCOMM         European Conditional Mouse Mutagenesis Program
 Flp         Site-specific recombinase from S.cerevisiae 
 Flpe         Flp, enhanced efficiency at 37 C = 
 Flpo         mammalian codon optimized, enhanced version of Flp
 FRT         Flp recombinase recognition target
 G         Gap repair (oligo)
 Gen         Gentamycin
 HA         Homology arm
 HGNC         HUGO Gene Nomenclature Committee
 Hprt         Hypoxantin phosphoribosyltransferase
 HR         Homologous Recombination
 HRP         Horseradish Peroxidase
 HUGO         Human Genome Organisation
 IGTC         International Gene Trap Consortium
 I-DCC         International Data Coordination Centre
 IKMC         International Knock Out Mouse Consortium
 IMMC         International Mouse Mutagenesis Consortium
 IMSR         International Mouse Strain Resource
 iPS cells         Induced pluripotent stem cells
 IRES         Internal Ribosome Entry Site
 KAN / KM         Kanamycin
 KO         Knock Out
 KOMP         Knock Out Mouse Project (USA)
 loxP         Locus of crossover (x) in P1 bacteriophage
 LR         Longe Range
 miRKO         micro RNA Knock Out
 MGH         Massachusetts General Hospital
 MGI         Mouse Genome Informatics
 MGNC         Mouse Genome Nomeclature Committee
 MGP         Mouse Genetics Program
 MM(R)RC         Mutant Mouse (Regional) Resource Centers
 MRC         Medical Resarch Council
 NCBI         National Center for Biotechnology Information
 neo         Neomycin/Kanamycin
 NIH         National Institute of Health
 NLS         Nuclear Localization Signal
 NMD         Nonsense-Mediated mRNA Decay
 NorKOMM         North American Conditional Mouse Mutagenesis project (Canada)
 OMIM         Online Mendelian Inheritance in Man (database)
 ori         Origin of replication
 pA         Polyadenylation signal
 PB         PiggyBAC (Transposon)
 PGK         promoter Phosphoglycerate kinase promoter
 PTC         Premature Termination Codon
 QC         Quality Control
 RE         Restriction Enzyme site
 RMCE         Recombinase-Mediaed Cassette Exchange
 RMGR         Recombinase-Mediaed Genomic Replacement
 rox         Dre specific recognition site
 RRS         Recombinase Recognition Site
 SB         SleepingBeauty (Transposon)
 SBP         Streptavidin Binding Protein/Peptide
 sm         Selectable marker
 SSR         Site Specific Recombinases
 Strep         Streptomycin
 TAP-tag         Tandem Affinity Purification
 TC         Targeting construct
 TET         Tetracycline
 TIGM         Texas Institute for Genomic Medicine
 U         Upstream (oligo)
 UCSC         University of California Santa Cruz
 UTR         Untranslated region
 VEGA         Vertebrate Genome Annotation
 WTAC         Wellcome Trust Advanced Courses
 WTSI         Wellcome Trust Sanger Institute