Genetic Drift: What It Is and Its Impact on Your Research
Name: Jens Fritsche, Ph.D. Date: Homburg, 24.10.2019
Presented by Charles River, exclusive distributor of JAX ™ Mice in Europe and parts of Asia Mission of The Jackson Laboratory™ “To discover precise genomic solutions for disease and empower the global biomedical community in the shared quest to improve human health.” Biomedical Research Genetics, genomics and biology of human disease
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JAX™ Mice | 3 Online Resources to Expedite Research JAX™ Mice Database www.jax.org/mouse-search
Mouse Genome Informatics www.informatics.jax.org
Mouse Phenome Database www.jax.org/phenome
Others, including: JAX-Clinical Knowledgebase Mouse Tumor Biology Database
JAX™ Technical Support | 4 Today’s Learning Goals
Recognize genetic background of your mouse strain
o Use proper nomenclature o Select appropriate controls
Implement strategies to reduce genetic drift and increase experimental reproducibility
JAX.org | 5 What is Genetic Drift? “The constant tendency of genes to evolve even in the absence of selective forces. Genetic drift is fueled by spontaneous neutral mutations that disappear or become fixed in a population at random”
o Lee Silver, “Mouse Genetics” Oxford University Press, 1995 www.informatics.jax.org/silverbook/
Genetic changes resulting from mistakes in meiosis or DNA repair
o Single base changes o Deletions o Duplications o Inversions
JAX.org | 6 Genetic Drift Promotes Diversity
muscular dystrophy Lama2dy-2J Phenotypic Diversity
Species Diversity
Data Diversity
JAX.org | 7 Visible Genetic Drift Coat Color Mutations
C57BL/6J-Aw-J/J B6(Cg)-Tyrc-2J/J (000051) (000058)
C57BL/6J (000664)
C57BL/6J-Lystbg-J/J C57BL/6J-KitW-v/J (000629) (000049)
JAX™ Mice | 8 How Rapidly Do Colonies Drift? “Visible” mutation example
Using spontaneous mutation rates in coat color genes,
-5 o Measured ~1.1 x 10 mutations/locus/gamete/gen. Assuming ~25,000 genes in mice,
-5 o (1.1 x 10 mutations/locus/gamete/gen.)*(25,000 loci) o 0.275 mutations/gamete/gen. o 1 mutations/3.64 gametes/gen. 1 phenotypic mutation arises every 1.8 generations o Likely underrepresents overall mutation rate due to visibility of mutation
Russell LB and Russell WL., 1996. PNAS PMID 8917546 Drake JW et al., 1998, Genetics PMID 9560386
JAX.org | 9 How Rapidly Do Colonies Drift?
Mice have a high rate of spontaneous mutation
Approx. 25% chance that new mutations will become fixed
New mutations in coding sequence become fixed every 6-9 generations
o (Assumptions: inbreeding; small breeding population)
JAX.org | 10 How Rapidly Do Colonies Drift? “Invisible” mutation example
Using whole genome sequencing of C57BL/6J,
o Measured 2 samples separated by 69 filial gens. Differences found
o 669 SNPs (~ 10/gen.) o 272/669 SNPs were in genetic coding & non-coding regions o 7/272 SNPs altered DNA coding sequence or RNA splicing ~1 “impactful” mutation every 10 generations o Likely underrepresents overall mutation rate because the analysis did not include non-SNP mutations (deletions, inversions, CNV changes).
JAX.org | 11 “Invisible” Genetic Drift in C57BL/6 Published example: Loss of presynaptic protein α-synuclein (Snca)
C57BL/6J Genomic DNA from The Jackson Laboratory Wild-type Snca
C57BL/6NCrl Mice from Charles River, Margate, UK Wild-type Snca
C57BL/6JOlaHsd Mice from Harlan, Bicester, UK Deletion of Snca – No visible phenotype but… SNCA protein: implicated in a range of neurodegenerative diseases; primary structural component of Lewy bodies found in Parkinson’s disease brains
Specht CG and Schoepfer R. 2001. BMC Neurosci 2:11. PMID:11591219
JAX® Mice | 12 Genetic Drift and Colony Size Small colonies are more vulnerable to fix a mutation
For any given mutation, = heterozygous mutation
JAX.org | 13 Genetic Drift and Colony Size Small colonies are more vulnerable to fix a mutation
WT Het Hom
JAX.org | 14 C57BL/6 Substrain Divergence Substrains: Branch of an inbred strain known or suspected to be genetically different from the parent colony.
JAX™ Mice | 15 Substrain Development
20 generations apart
JAX® Mice | 16 Know Your Substrain Use Proper Nomenclature
C57BL/6J Parent strain
Substrain designation C57BL/6NJ NIH (N) By (Dr. Bailey)
C57BL/6NCrl Laboratory maintaining the strain Jackson (J) Crl (Charles River Laboratories)
C57BL/6ByJ
Institute for Laboratory Animal Research (ILAR) Lab Codes http://dels.nas.edu/global/ilar/Lab-Codes
JAX™ Mice | 17 C57BL/6 Publications
Complete nomenclature benefits everyone!
Based on October, 2018 PubMed citations search (without limits)
Nomenclature for Mouse Strains | 18 But Aren’t All B6 Mice the Same? C57BL/6 substrains are not the same! They differ genetically
o Single Nucleotide Polymorphisms (SNPs) o Insertions & deletions (Indels) o Copy number variations (CNVs) o Spontaneous mutations
JAX™ Mice | 20 Genetic Differences Translate into Phenotypic Differences
Neurobiology
o Retinal degeneration, Case #1 Metabolism
o Response to high-fat diet, Case #2 Immunology
o Sensitivity to infection, Case #3 o Immune phenotypes, Case #4 And more…
JAX™ Mice | 21 “Invisible” Genetic Drift in C57BL/6 Case Study #1: Retinal degeneration in C57BL/6N substrains
Crb1 (crumbs-like 1) Localized to Muller cells and photoreceptor (PC) inner segments Mutations in CRB1 associated with retinal diseases in humans Retinitis pigmentosa o http://crfb.univ-mrs.fr/Crumbs/section/en/CRB1_function/105 o Leber congenital amaurosis Crb1rd8 Single base deletion Shorter PC inner & outer segments as early as two weeks Progressive, spotty retinal degeneration
Mehallow AK et al. 2003. Hum Mol Gen 12(17):2179-2189. PMID:12915475
JAX™ Mice | 22 “Invisible” Genetic Drift in C57BL/6 Case Study #1: Retinal degeneration in C57BL/6N substrains
C57BL/6J: Crb1 wild-type C57BL/6N: Crb1rd8/Crb1rd8
C57BL/6N – For C57BL/6N – Rev C57BL/6J – For C57BL/6J – Rev
ALL C57BL/6N substrains are Crb1rd8/Crb1rd8
HSD (Harlan) CRL (Charles River) rd8 DCT (Frederick Nat’l Lab) wild-type TAC (Taconic)
Mattapallil, MJ et al. 2012. Invest Ophthalmol Vis Sci PMID 22447858
JAX™ Mice | 23 Metabolic Differences (DIO) Case Study #2: Response to high-fat diet in C57BL/6
C57BL/6J (000664) vs C57BL/6NJ (005304) Mice fed a 60 kcal% high fat diet
Nicholson, A et al. 2010. Obesity 18(10): 1902-1905. PMID: 20057372
JAX™ Mice | 24 Metabolic Differences (DIO) Case Study #2: Response to high-fat diet in C57BL/6
C57BL/6J (000664) vs C57BL/6NJ (005304) 2 wks on HFD 14 wks on HFD
Glucose Tolerance Test Measures ability of mice to clear glucose from blood Both B6J and B6NJ mice have severely impaired glucose tolerance
Nicholson, A et al. 2010. Obesity 18(10): 1902-1905. PMID: 20057372
JAX™ Mice | 25 Immunological Differences Case Study #3: Sensitivity to infection
1952
C3H/HeJ C3H/HeOuJ (000659) (000635)
+LPS +LPS
Endotoxin resistant Endotoxin sensitive Tlr4Lps-d (1958-1965) Tlr4 wild-type
Sultzer BM. 1968. Nature PMID 4877918 Watson J et al. 1978. J Immunol PMID 202651 Poltarak A et al. 1998. Blood Cells Mol Dis PMID 10087992 Poltarak A et al. 1998. Science PMID 9851930
JAX™ Mice | 26 Immunological Differences Case Study #4: Dock2 CNV in C57BL6/NHsd affects immune phenotypes
CD8+ MP MZB Live, Live, CD3+, CD8+ CD19+, IgDlo IgMhi
C57BL/6J
C57BL/6NJ
C57BL/6NHsd Increased CD8+ memory T cells Loss of marginal zone B cells
Mahajan, V et al. 2016. Cell Reports PMID: 27210752
JAX™ Mice | 27 More Published Examples
JAX™ Mice | 28 Considerations for Control Selection
Congenic Strains* o Littermates (het x het, het x wt, or hemi x wt mating scheme) Wild type or heterozygous for mutant gene or allele Non-carriers of transgene Can also use non-littermate controls from the colony o Inbred (hom x hom mating) Match background mutant is on (including substrain) ? Mixed Background (B6J and B6N) o Littermates Wild type or heterozygous for mutant gene or allele Non-carriers of transgene Can also use non-littermate controls from the colony
* Congenic strains have been crossed more than 10 generations to inbred strain. Acceptable to use inbred as control after N5
JAX™ Mice | 29 Selecting Proper Controls Case Study # 5: C57BL/6 control selection Influence of Mapk9 (Jnk2) on acetaminophen-induced liver injury (AILI)
Bourdi M et al. 2011. Chem Res Toxicol PMID 21557537
JAX™ Mice | 30 Selecting Proper Controls Experimental conclusions may be in opposition Effects of Mapk9 (Jnk2) on acetaminophen-induced liver injury (AILI)
Bourdi M et al. 2011. Chem Res Toxicol PMID 21557537
JAX™ Mice | 31 Selecting Proper Controls Experimental conclusions may be in opposition Effects of Mapk9 (Jnk2) on acetaminophen-induced liver injury (AILI)
Bourdi M et al. 2011. Chem Res Toxicol PMID 21557537
JAX™ Mice | 32 Genetic Drift and Substrains
Spontaneous mutations can be overt or hidden o only apparent by physiological assay
Substrains can vary significantly genetically and phenotypically
Know what substrain backgrounds your strains are, and use the proper control
JAX.org | 34 How to Detect Genetic Drift
Comparing whole genome sequence data
o Single Nucleotide Polymorphism (SNP) scans won’t do it Look for phenotypic differences
image
JAX.org | 35 Minimizing Genetic Drift
Genetic change can’t be stopped, but it can be slowed down!
Maintain pedigrees and detailed colony records
JAX™ Mice |
JAX.org | 36 Maintaining a Pedigreed Colony
Single Established Colony - any strain type sister-brother mating only!
Mutations become fixed more rapidly in sister-brother pedigrees • More easily identified/more easily removed
Breeding & Rederivation Services | 37 Minimizing Genetic Drift Genetic change can’t be stopped, but it can be slowed down!
Maintain pedigrees lines and detailed colony records Watch for phenotypic changes in mutants and controls Refresh breeders frequently (~10 generations) Avoid selection pressure Verify genetic background with genome scanning Cryopreserve unique strains
JAX.org | 38 The Jackson Laboratory’s Genetic Stability Program (GSP)
JAX Genetic Stability Program | 40 Genetic Stability Program Works!
Whole genome sequencing on C57BL/6J genomic DNA 1984 @ F154 (pre GSP) } 69 gen. 2003 @ F223 (GSP “Eve”) } 7 gen. 2012 @ F230 (post-GSP) Evaluated high quality single nucleotide variants (SNPs)
Constant mutation rate in pre- & post-GSP period Mutations accumulate more slowly post-GSP
JAX Genetic Stability Program | 41 Summary
Spontaneous mutations occur frequently and can be overt or hidden o Implement breeding and colony maintenance strategies that minimize genetic drift Substrains can vary significantly genetically and phenotypically
o Know the substrain that you use, and use the proper control
JAX.org | 42 Charles River is the Thank you! Exclusive Distributor of JAX™ Mice in Europe and parts of Asia.*
Contact Charles River: [email protected]
*Europe: Albania, Austria, Belgium, Bosnia- Herzegovina, Bulgaria, Croatia, Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Japan, Luxembourg, JAX™ Mice, Clinical & Macedonia, Montenegro, Netherlands, Norway, Research Services Poland, Portugal, Serbia, Slovenia, Slovakia, Spain, Switzerland, Sweden, and the United Kingdom www.jax.org [email protected] *Asia: Japan, Korea, Taiwan
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