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Technical Sheet Marker-Assisted Accelerated Backcrossing (MAX-BAX®) Time is of the essence when creating research models, and marker-assisted accelerated backcrossing has proven valuable in expediting the process of creating congenic strains. Charles River, the industry leader in rodent research models, offers MAX-BAX® for the directed selection of individuals/breeders with a preferred genetic background. By backcrossing selectively rather than randomly, you can significantly decrease the number of backcross generations needed to produce a congenic strain, thereby producing a research model faster, while simultaneously reducing your facility, equipment and personnel costs. MAX-BAX® Technology Congenic strains are widely used in biomedical research because they reduce genetic variability and provide insight into the contribution of genetic background to phenotype. Congenic strains are identical at all genetic loci except for one; that differing locus is usually the transgene or knockout region of interest. Utilizing traditional, random backcrossing methods, it takes 10 generations (upwards of 2.5 years) to produce a congenic strain. Selectively breeding individuals containing more of the recipient genome from each generation allows for accelerated congenic strain production (Table 1).Those animals carrying the locus of interest with the highest percentage of recipient versus donor strain DNA are preferentially bred. Genetic markers mapped to specific locations on each chromosome are used to evaluate strain-specific genomic polymorphism. Our mouse single nucleotide polymorphism (SNP) panel, composed of 384 carefully selected SNP markers, was designed to maximize polymorphism between common inbred strains and provide even coverage of the mouse genome. Markers are spaced at approximately 7 Mbp intervals, and about half of the markers will be polymorphic between any inbred or outbred strains, allowing the same panel to be used for any donor and recipient strains. The testing for the 384 SNP marker panel is performed on a microarray platform using robust fluorescence-based SNP genotyping assays (Figure 1). Results yield a defined analysis of the genome in question, and a preferred breeding rank is determined for all test individuals. The entire genome is analyzed at each generation, which may make unexpected genetic variation or breeding errors easier to detect. The 384 mouse SNP panel complements our other mouse and rat genetic background panels for inbred and outbred lines. These include our 32 marker SNP panels for routine genetic quality control and custom microsatellite panels for speed congenics, mapping, fine mapping, and substrain or background strain characterization. TABLE 1. Congenic Strain Production Strategies Traditional Backcross Speed Backcross Generation Recipient Genome Generation Recipient Genome F1 50.00% F1 50.00% N2 75.00% N2 ~80.00% N3 87.50% N3 ~94.00% N4 93.75% N4 ~99.00% N5 96.88% N5 ~100.00% N6 98.44% N7 99.22% N8 99.61% N9 99.81% N10 99.90% [email protected] www.criver.com © 2013, Charles River Laboratories International, Inc. technical sheet Figure 1. SNP Genotyping Data forData 2 Markers FIGURE 1. SNP Genotyping for 2 SNP rs13476621 Chromosome 2, 87.19 Mb Markers 2. Two to three animals having the highest percentage of recipient strain contribution, as determined by MAX-BAX® analysis, should be mated to recipient strain animals The Figure 1 showsaim the fluorescence is to produce about 10 heterozygotes for subsequent data for 2 SNP markers. Each point represents the genotype of strain assessment. These animals will be background one animal for that marker. Blue and red pointsdesignated are animals that the N2 generation. SNP rs13480740 Chromosome 10, 103.52 Mb FAM Intensity FAM Intensity 8699 10135 6862 7939 5025 5742 3189 3546 are homozygous for one of the two possible alleles, while green points 3. thatThis process of are animals are heterozygous for selected breeding will be repeated at both possible alleles. Each animal each generation typically to N5, at which point congenic is assayed for 384 markers, and its genetic profile is compared to that of individuals should be obtained. the recipient strain to identify animals with the highest amount of the desired background. 4. The time frame, assuming no problems with breeding or health, etc., will be approximately 98 days per generation or 490 days to complete the project (1.3 years) Figure 1. shows the fluorescence data for 2 SNP markers. The testing for the 384 SNP marker panel is performed 2. Two to three animals havingConventional the highest percentage backcrossing takes upwards of 2.5 years to Each point represents therobust genotype of one animal for of that on a microarray platform using fluorescence-based recipient strain contribution, as determined by produce the desired congenic strain. SNP genotyping assays 1). are Results yield a that defined MAX-BAX® analysis, should be mated to recipient strain marker. Blue and red(Figure points animals are homozygous analysis of the genome in question, and a preferred animals The aim is to produce about 10 heterozygotes for one of the two possible while green points are breeding rank is determined for allalleles, test individuals. for subsequent background strain assessment. These Test Results and Sample Submission The entire that genome analyzed at eachfor generation, animals the N2 generation. animals areisheterozygous both possible alleles. Eachwill be designated ® Results from MAX-BAX analysis are typically reported in one which may make unexpected genetic variation or animal is assayed for 384 markers, and its genetic profile is 3. This process of selected breeding will be repeated breeding errors easier to detect. week. Tail snips should be collected and immersed in 70% ethanol compared to that of the recipient strain to identify animals at each generation typically to N5, at which point and refrigerated (4°C) prior to shipment. Other tissues, such as ear The 384 mouse SNP panel complements our other mouse congenic individuals should be obtained. with the highest amount of the desired background. and rat genetic background panels for inbred and outbred punches or toe clips may also be used. Please contact Charles 4. The time frame, assuming no problems with breeding lines. include 32 marker SNPindividual panels for routine As theThese animals areourbackcrossed, loci, and eventually Laboratories priorper to shipment. or health, etc., will River be approximately 98 days genetic quality control and custom microsatellite panels entire chromosomes, will become fixed and for the recipient allele and or 490 days to complete the project generation for speed congenics, mapping, fine mapping, (1.3 years) backcrossing takes upwards substrain background strain characterization. no longerorrequire monitoring. This allows us to custom design a Conventional Please contact Charles River when planning your MAX-BAX® of 2.5 years to produce the desired congenic strain. panel for every subsequent backcross generation, saving both program or with other questions regarding our genetic testing MAX-BAX® General Principles Test Results and Sample Submission time and money in the process. services of knockout and transgenic rodents. 2239 3401 4564 5726 6888 8050 921 2208 2953 3698 VIN Intensity 4443 5188 5933 6678 VIN Intensity While the absolute parameters of every MAX-BAX® project are unique, we have listed some general principles that ® a road map to plan can be used as MAX-BAX General Principles a MAX-BAX® program. ® Results from MAX-BAX® analysis are typically reported in one week. Tail snips should be collected and immersed in 70% ethanol and refrigerated (4°C) prior to shipment. Other While the absolute parameters of every MAX-BAX project are such as ear punches or toe clips may also be used. tissues, contact Charles River Laboratories prior 1. If the initial are already a defined background, unique, we animals have listed someongeneral principles that canPlease be used to shipment. the F1 progeny (of the founder animal and ® a mate of the asrecipient a road strain) map to plan a MAX-BAX program. will all be 50% recipient strain, so there is Please contact Charles River when planning no advantage in performing genetic analysis at this point 1. If the initial animals are already on a defined background, your MAX-BAX® program or with other questions If the founder animal is homozygous for the gene of regarding the F1 progeny (of the founder animal and a mate of the our genetic testing services of knockout interest, the progeny are obligate heterozygotes, and transgenic rodents. genotyping for thestrain) gene ofwill interest also not necessary recipient all beis 50% recipient strain, soand there for this generation. If the initial animals are on a hybrid is no advantage in performing genetic analysis at this background, and the desired recipient strain is one of the ® strain point contributing this background, MAX-BAX If thetofounder animal is homozygous for the gene genotyping of the F1 generation can identify animals with of interest, the progeny are obligate heterozygotes, and a higher-than average percentage of the recipient strain. genotyping for the gene of interest is also not necessary for this generation. If the initial animals are on a hybrid background, and the desired recipient strain is one of the strain contributing to this background, MAX-BAX® genotyping of the F1 generation can identify animals with a higher-than average percentage of the recipient strain. [email protected] www.criver.com © 2011, Charles River Laboratories International, Inc. [email protected] www.criver.com © 2013, Charles River Laboratories International, Inc.