Genetic Quality of Laboratory Animals The ABC of Animal Experiments 2008 NLAC Kristiina Raatesalmi, Ph. D.
Properties of animals suitable for research <ul><li>free of adventitious infections that cause infections that cause disea...
Terms <ul><li>Visible or otherwise measurable properties are called  phenotype </li></ul><ul><li>Genetic factors responsib...
<ul><li>Genes, although stable, occasionally change  </li></ul><ul><li>mutation </li></ul><ul><li>The organism carrying th...
<ul><li>Alternative forms of a gene that arise as a results of mutations are called  alleles </li></ul><ul><li>When both g...
Relationship between alleles <ul><li>Dominant/Resessive </li></ul><ul><li>the phenotype is determined by the dominant alle...
Linkage <ul><li>Unlinked genes </li></ul><ul><li>located in different chromosomes </li></ul><ul><li>assort independently d...
Mutations  continues <ul><li>Any modification which affects DNA (e.g. deletion,  insertion...) </li></ul><ul><li>spontaneo...
Abnormalities may be associated with a visible shange <ul><li>hairless  (Flanagan 1966; Gen. Res. 8: 295) </li></ul><ul><l...
nude-mouse
Changes may not have effect on appearance <ul><li>Selection </li></ul><ul><li>SCID  mouse  (Bosma et al. 1981: Nature 301:...
Categories of characterized animals <ul><li>􀂄 ( Spontaneous mutants ) </li></ul><ul><li>Outbred </li></ul><ul><li>􀂄 Random...
Definitions <ul><li>Outbred (stock) </li></ul><ul><li>genetically variable stock, which is maintained so that </li></ul><u...
Characteristics of outbred stock <ul><li>heterozygosity undefined </li></ul><ul><li>􀂄 genetic profile unknown </li></ul><u...
Outbred nomenclature <ul><li>􀂄 first letters before colon (:) reveal the place where the  stock is maintained (Kuopio) </l...
Outbred stocks <ul><li>Goal of management system: </li></ul><ul><li>􀂄 maintain a maximum genetic variability (polymorphism...
Genetic changes in outbred stock <ul><li>􀂄  genetic contamination or immigration </li></ul><ul><li>􀂄  mutation </li></ul><...
<ul><li>lethal mutations are not transmitted to the next generation </li></ul><ul><li>some may be interesting to detect an...
Outbred stock: Reference colony <ul><li>Long term maintenance of genetic variability / </li></ul><ul><li>polymorphism </li...
Robertson´s 4-unit mating scheme <ul><li>Male from Female from New unit </li></ul><ul><li>U1 U2 U1 </li></ul><ul><li>U3 U4...
Random bred, non-inbred, non-outbred <ul><li>􀂄  Inbred strains not maintained by continued </li></ul><ul><li>inbreeding (b...
Characteristics of inbred strains <ul><li>At least 20 consecutive generations of brother x sister matings (inbreeding coef...
Inbred strains <ul><li>Are normally murine </li></ul><ul><li>short life cycle </li></ul><ul><li>large number of offsprings...
The first genetically defined inbred rodent strain <ul><li>progenitor of DBA-strain </li></ul><ul><li>produced by Clarence...
Inbred rodent production Pedigree nucleus colony Gnotobiotic nucleus colony PNC EC Non-pedigree expansion colony PC Random...
Inbred strain <ul><li>Problems in production: </li></ul><ul><li>􀂄  inbreeding depression may appear after 6-7 </li></ul><u...
Properties of inbred strains <ul><li>􀂄 isogenicity </li></ul><ul><li>􀂄  homozygosity </li></ul><ul><li>􀂄  phenotypic unifo...
Commonly used inbred strains of mice <ul><li>Strain Abbrevi- Research </li></ul><ul><li>ation use </li></ul><ul><li>AKR  A...
Spontaneous pathology of some inbred strains <ul><li>􀂄  DBA – Mammary tumors </li></ul><ul><li>􀂄  C3H – Mammary tumors </l...
Details of nomenclature <ul><li>􀂄 The Jackson Laboratory web site </li></ul><ul><li>www.informatics.jax.org/nomen   </li><...
Backcross <ul><li>a crossing of a heterozygous organism and a homozygote </li></ul><ul><li>commonly refers to the transfer...
Testing linkage <ul><li>aabb x AaBb (dihybrid backcrossed to the double recessive parental strain) </li></ul><ul><li>linka...
F1 hybrid <ul><li>Inbred strain  X   Inbred strain </li></ul><ul><li>A  B </li></ul><ul><li>  A-B hybrid  </li></ul><ul><l...
Advantages of F1-hybrid <ul><li>Vs. inbred  </li></ul><ul><li>heterosis (hybrid vigour) </li></ul><ul><li>phenotype stable...
Isogenicity <ul><li>All individuals within an inbred or F1 hybrid strain are genetically identical  </li></ul>AA aa Aa X
<ul><li>Coisogenic strains </li></ul><ul><li>-two inbred starins that differ by a single mutant gene, but otherwise are ge...
<ul><li>this is done by crossing the background inbred strain with the gene donor; repeated </li></ul><ul><li>after 8 gene...
Nomenclature <ul><li>Coisogenic strains </li></ul><ul><li>background strain, slash, symbol of the differential allele; C57...
Recombinant inbred (RI) and recombinant congenic (RC) strains <ul><li>RI strains </li></ul><ul><li>inbred strains derived ...
Subline divergence of inbred animals <ul><li>genetic contamination </li></ul><ul><li>residual heterozygosity </li></ul><ul...
Genetic quality control for inbred/F1 hybrid rodents <ul><li>Preserve isogenicity by preventing and detecting subline dive...
Detection of genetic contamination <ul><li>substantial increase in litter size </li></ul><ul><ul><li>sign of heterosis </l...
Genetic monitoring <ul><li>immunogenetic monitoring </li></ul><ul><ul><li>skin grafting </li></ul></ul><ul><ul><li>serum p...
Choice of markers <ul><li>simple inheritance </li></ul><ul><li>phenotype corresponds to genotype </li></ul><ul><li>found o...
Genetic quality control <ul><li>PCR (polymerase chain reaction) </li></ul><ul><li>􀂄  primers are commercially available e....
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C.GenQuality.ppt

  1. 1. Genetic Quality of Laboratory Animals The ABC of Animal Experiments 2008 NLAC Kristiina Raatesalmi, Ph. D.
  2. 2. Properties of animals suitable for research <ul><li>free of adventitious infections that cause infections that cause disease or affect research </li></ul><ul><li>express stable, identifiable, gentically determined charasteristics </li></ul>
  3. 3. Terms <ul><li>Visible or otherwise measurable properties are called phenotype </li></ul><ul><li>Genetic factors responsible for creating the phenotype are called genotype </li></ul>AaBBCc
  4. 4. <ul><li>Genes, although stable, occasionally change </li></ul><ul><li>mutation </li></ul><ul><li>The organism carrying the altered gene is called a mutant </li></ul><ul><li>The organism carrying the normal gene is called a wild type </li></ul>
  5. 5. <ul><li>Alternative forms of a gene that arise as a results of mutations are called alleles </li></ul><ul><li>When both gene copies are </li></ul><ul><li>identical, the organism is called a homozygote (AA) </li></ul><ul><li>When the gene copies are </li></ul><ul><li>different, the organism is called a heterozygote (Aa) </li></ul>
  6. 6. Relationship between alleles <ul><li>Dominant/Resessive </li></ul><ul><li>the phenotype is determined by the dominant allele so the appearance of the heterozygote is indistinguishable from that of the homozygous dominant parent </li></ul><ul><li>Partial dominance </li></ul><ul><li>the heterozygote is intermediate between the two homozygotes </li></ul><ul><li>Codominance </li></ul><ul><li>both alleles contribute equally to the phenotype </li></ul>
  7. 7. Linkage <ul><li>Unlinked genes </li></ul><ul><li>located in different chromosomes </li></ul><ul><li>assort independently during meiosis (Mendel´s second law) </li></ul><ul><li>Linked genes </li></ul><ul><li>located on the same chromosomes tend to be inherited together </li></ul>
  8. 8. Mutations continues <ul><li>Any modification which affects DNA (e.g. deletion, insertion...) </li></ul><ul><li>spontaneous mutations (coat color, obesity, nude) </li></ul><ul><li>mutations by chemicals, radiation e.t.c. </li></ul><ul><li>They modify the gene sequence and can generate a new allelic form which can lead to a new phenotype. </li></ul><ul><li>􀂄 low frequency (10 -8 to 10 -5 per germ cell) </li></ul><ul><li>􀂄 most of them affect non -coding DNA sequence </li></ul><ul><li>􀂄 most of them are recessive and are detected much later on homozygous individuals </li></ul><ul><li>􀂄 </li></ul>
  9. 9. Abnormalities may be associated with a visible shange <ul><li>hairless (Flanagan 1966; Gen. Res. 8: 295) </li></ul><ul><li>highly susceptible to infectious disease </li></ul><ul><li>athymic (nude) (Pantelouris 1968; Nature 217: 370) </li></ul><ul><li>deficient in T-cell mediated immunity </li></ul><ul><li>accept tumor grafts </li></ul><ul><li>particularly useful for cancer research </li></ul>
  10. 10. nude-mouse
  11. 11. Changes may not have effect on appearance <ul><li>Selection </li></ul><ul><li>SCID mouse (Bosma et al. 1981: Nature 301: 527) </li></ul><ul><li>absence of T and B lymphocytes </li></ul><ul><li>very useful of studying HIV </li></ul><ul><li>SHR rat (WIST:Kyoto; Okamoto and Aoki 1963: Jpn. Circ. J. 27: 282) </li></ul><ul><li>spontaneously hypertensive </li></ul><ul><li>AA/ANA rat (Eriksson 1968: Science 159: 739) </li></ul>
  12. 12. Categories of characterized animals <ul><li>􀂄 ( Spontaneous mutants ) </li></ul><ul><li>Outbred </li></ul><ul><li>􀂄 Random-bred, non-outbred, non-inbred </li></ul><ul><li>􀂄 Inbred </li></ul><ul><li>􀂄 Recombinant inbred </li></ul><ul><li>􀂄 Coisogenic and congenic; mutants </li></ul><ul><li>􀂄 Segregating inbred </li></ul><ul><li>􀂄 Consomic </li></ul><ul><li>􀂄 F1 hybrids </li></ul>
  13. 13. Definitions <ul><li>Outbred (stock) </li></ul><ul><li>genetically variable stock, which is maintained so that </li></ul><ul><li>there is less than 1% inbreeding per generation (mating by unrelated individuals) </li></ul><ul><li>Inbred (strain) </li></ul><ul><li>20 consecutive generations with brother-sister (full-sib) mating, or mating offspring with it’s younger parent </li></ul><ul><li>inbreeding coefficient F=98,6 % (the measure of the reduction in heterozygosity) </li></ul><ul><li>(1,4 % recidual heterozygosity > subline divergence) </li></ul>
  14. 14. Characteristics of outbred stock <ul><li>heterozygosity undefined </li></ul><ul><li>􀂄 genetic profile unknown </li></ul><ul><li>􀂄 random-mated (or rotation), closed colonies </li></ul><ul><li>􀂄 high reproductive performance </li></ul><ul><li>􀂄 high vigour </li></ul><ul><li>􀂄 phenotypically variable </li></ul><ul><li>􀂄 no “right” genetic characterization </li></ul>
  15. 15. Outbred nomenclature <ul><li>􀂄 first letters before colon (:) reveal the place where the stock is maintained (Kuopio) </li></ul><ul><li>􀂄 last letters show the stock’s name (WISTAR) </li></ul><ul><li>􀂄 name given after three generations Kuo:WIST </li></ul>
  16. 16. Outbred stocks <ul><li>Goal of management system: </li></ul><ul><li>􀂄 maintain a maximum genetic variability (polymorphism) </li></ul><ul><li>􀂄 breed “homogenous” individuals within a same stock </li></ul><ul><li>maintain stable allelic frequencies from generation to generation </li></ul><ul><li>minimize the increase of inbreeding from generation to generation </li></ul>
  17. 17. Genetic changes in outbred stock <ul><li>􀂄 genetic contamination or immigration </li></ul><ul><li>􀂄 mutation </li></ul><ul><li>􀂄 directional selection </li></ul><ul><li>􀂄 random drift </li></ul>
  18. 18. <ul><li>lethal mutations are not transmitted to the next generation </li></ul><ul><li>some may be interesting to detect and to preserve to create pathological models </li></ul>
  19. 19. Outbred stock: Reference colony <ul><li>Long term maintenance of genetic variability / </li></ul><ul><li>polymorphism </li></ul><ul><li>closed colony, no introduction of new breeders </li></ul><ul><li>minimum 120 permanent breeding pairs </li></ul><ul><li>fixed reproduction life : 30 weeks </li></ul><ul><li>mating scheme : 4-unit-Robertson's scheme </li></ul><ul><li>min 84 % of pairs contribute to the next generation </li></ul><ul><li>no selection to increase productivity </li></ul><ul><li>future breeders from 4 th litter, max 3 female breeders per pair </li></ul><ul><li>􀂄 </li></ul>
  20. 20. Robertson´s 4-unit mating scheme <ul><li>Male from Female from New unit </li></ul><ul><li>U1 U2 U1 </li></ul><ul><li>U3 U4 U2 </li></ul><ul><li>U2 U1 U3 </li></ul><ul><li>U4 U3 U4 </li></ul>
  21. 21. Random bred, non-inbred, non-outbred <ul><li>􀂄 Inbred strains not maintained by continued </li></ul><ul><li>inbreeding (brother x sister matings) </li></ul><ul><li>􀂄 Outbred stocks maintained in too small breeding </li></ul><ul><ul><li>colonies </li></ul></ul>
  22. 22. Characteristics of inbred strains <ul><li>At least 20 consecutive generations of brother x sister matings (inbreeding coefficient 98,6 %) </li></ul><ul><li>&quot;clone&quot; </li></ul><ul><li>􀂄 genetic profiles known </li></ul>
  23. 23. Inbred strains <ul><li>Are normally murine </li></ul><ul><li>short life cycle </li></ul><ul><li>large number of offsprings </li></ul><ul><li>small </li></ul><ul><li>need little space </li></ul><ul><li>small costs </li></ul>
  24. 24. The first genetically defined inbred rodent strain <ul><li>progenitor of DBA-strain </li></ul><ul><li>produced by Clarence C. Little in 1909 </li></ul><ul><li>selection with specific coat colours </li></ul><ul><li>today´s DBA strains are mainly used to study the effect ovat hereditary factors on the development of cancer </li></ul>
  25. 25. Inbred rodent production Pedigree nucleus colony Gnotobiotic nucleus colony PNC EC Non-pedigree expansion colony PC Random-mated production colony Inbred strains available for research
  26. 26. Inbred strain <ul><li>Problems in production: </li></ul><ul><li>􀂄 inbreeding depression may appear after 6-7 </li></ul><ul><li>generations </li></ul><ul><li>􀂄 reduced growth and deformities </li></ul><ul><li>The specific inheritable characteristics may </li></ul><ul><li>disappear somewhere along the line </li></ul>
  27. 27. Properties of inbred strains <ul><li>􀂄 isogenicity </li></ul><ul><li>􀂄 homozygosity </li></ul><ul><li>􀂄 phenotypic uniformity </li></ul><ul><li>􀂄 long term stability </li></ul><ul><li>􀂄 individuality </li></ul><ul><li>􀂄 international distribution </li></ul><ul><li>􀂄 identifiably </li></ul><ul><li>􀂄 sensitivity </li></ul>
  28. 28. Commonly used inbred strains of mice <ul><li>Strain Abbrevi- Research </li></ul><ul><li>ation use </li></ul><ul><li>AKR AK Leukemia model </li></ul><ul><li>BALB/c C Pristane priming, toxicol </li></ul><ul><li>C3H C3 Tumor model </li></ul><ul><li>C57BL/6 B6 Genetic background, </li></ul><ul><li>superovulation, general purpose model </li></ul><ul><li>DBA/2 D2 Tumor model </li></ul><ul><li>NZB ZB Autoimmune disease mo </li></ul>
  29. 29. Spontaneous pathology of some inbred strains <ul><li>􀂄 DBA – Mammary tumors </li></ul><ul><li>􀂄 C3H – Mammary tumors </li></ul><ul><li>􀂄 NZB – Autoimmune anemia </li></ul><ul><li>􀂄 BALB/C, DBA/ - Hypertension and/or cardiac </li></ul><ul><li> abnormalities </li></ul><ul><li>􀂄 129/Sv – Testicular teratomas </li></ul><ul><li>􀂄 AKR – Leukemia </li></ul>
  30. 30. Details of nomenclature <ul><li>􀂄 The Jackson Laboratory web site </li></ul><ul><li>www.informatics.jax.org/nomen </li></ul><ul><li>􀂄 The Rat Genome Database web site </li></ul><ul><li>www.rgd.mcw.edu </li></ul>
  31. 31. Backcross <ul><li>a crossing of a heterozygous organism and a homozygote </li></ul><ul><li>commonly refers to the transfer of a particular gene(s) from one background strain/stock to an inbred strain via multigenerational matings to the desired strain </li></ul>
  32. 32. Testing linkage <ul><li>aabb x AaBb (dihybrid backcrossed to the double recessive parental strain) </li></ul><ul><li>linkage indicated when less than half the offspring express recombinant phenotypes </li></ul><ul><li>In the case of linked genes recombinant genotypes are formed by crossing over between homologous chromosomes during meiosis </li></ul>
  33. 33. F1 hybrid <ul><li>Inbred strain X Inbred strain </li></ul><ul><li>A B </li></ul><ul><li> A-B hybrid </li></ul><ul><li>Heterozygous at all alleles which differ in parental strains </li></ul>
  34. 34. Advantages of F1-hybrid <ul><li>Vs. inbred </li></ul><ul><li>heterosis (hybrid vigour) </li></ul><ul><li>phenotype stable, less susceptibility to: </li></ul><ul><ul><li>environmental fluctuations </li></ul></ul><ul><ul><li>recessive mutations􀂄 </li></ul></ul><ul><li>Vs. outbred </li></ul><ul><li>isogenicity </li></ul><ul><li>long-term genetic stability </li></ul><ul><li>identifiability </li></ul><ul><li>phenotypic uniformity </li></ul><ul><li>accept graft from either parental strain </li></ul>
  35. 35. Isogenicity <ul><li>All individuals within an inbred or F1 hybrid strain are genetically identical </li></ul>AA aa Aa X
  36. 36. <ul><li>Coisogenic strains </li></ul><ul><li>-two inbred starins that differ by a single mutant gene, but otherwise are genetically the same </li></ul><ul><li>Congenic strains </li></ul><ul><li>-similar to coisogenic strains in that they primarily differ at a single gene </li></ul><ul><li>-however, while coisogenic strains arise spontaneously as a result of mutation, congenic strains are produced by introducing a gene from a donor strain or stock onto an inbred background </li></ul>
  37. 37. <ul><li>this is done by crossing the background inbred strain with the gene donor; repeated </li></ul><ul><li>after 8 generations the likelihood than an unselected, unlinked gene will be homozygous for the allele carried by the background strain is 99,3 % </li></ul><ul><li>passanger genes (genes remaining from the donor) </li></ul><ul><li>congenic after 12 generations </li></ul><ul><li>heterozygous intercrossed > homozygotes </li></ul>
  38. 38. Nomenclature <ul><li>Coisogenic strains </li></ul><ul><li>background strain, slash, symbol of the differential allele; C57BL/6JLepob. </li></ul><ul><li>heterozygous state is indicated by a +sign, slash, and the gene symbol; C3H/N-+/Wv </li></ul><ul><li>Congenic strains </li></ul><ul><li>􀂄 background strain (often abbreviated), period, the donor strain designation, hyphen and the allele designation; B10.129-H12b. </li></ul>
  39. 39. Recombinant inbred (RI) and recombinant congenic (RC) strains <ul><li>RI strains </li></ul><ul><li>inbred strains derived from full-sib matings of the F2 generation of a cross between two progenitor strains to study genetic linkage </li></ul><ul><li>RC strains similar system </li></ul>
  40. 40. Subline divergence of inbred animals <ul><li>genetic contamination </li></ul><ul><li>residual heterozygosity </li></ul><ul><ul><li>genetic drift </li></ul></ul><ul><li>genetic mutation </li></ul>?????
  41. 41. Genetic quality control for inbred/F1 hybrid rodents <ul><li>Preserve isogenicity by preventing and detecting subline divergence, especially its most important cause, </li></ul><ul><li>genetic contamination , </li></ul><ul><li>through </li></ul><ul><li>colony management </li></ul><ul><ul><li>colony structure </li></ul></ul><ul><ul><li>detailed breeding records </li></ul></ul><ul><ul><li>separation of common coat colors </li></ul></ul><ul><ul><li>policy: euthanize escaped animals </li></ul></ul><ul><li>genetic monitoring </li></ul>
  42. 42. Detection of genetic contamination <ul><li>substantial increase in litter size </li></ul><ul><ul><li>sign of heterosis </li></ul></ul><ul><li>morphological change (e.g. coat color, measurement of mandible) </li></ul>
  43. 43. Genetic monitoring <ul><li>immunogenetic monitoring </li></ul><ul><ul><li>skin grafting </li></ul></ul><ul><ul><li>serum protein allotypes </li></ul></ul><ul><li>biochemical </li></ul><ul><ul><li>allozymes </li></ul></ul><ul><li>molecular DNA polymorphism </li></ul><ul><ul><li>PCR </li></ul></ul><ul><ul><li>Southern blotting </li></ul></ul>
  44. 44. Choice of markers <ul><li>simple inheritance </li></ul><ul><li>phenotype corresponds to genotype </li></ul><ul><li>found on chromosomes (linkage groups) </li></ul><ul><li>codominant alleles </li></ul>
  45. 45. Genetic quality control <ul><li>PCR (polymerase chain reaction) </li></ul><ul><li>􀂄 primers are commercially available e. g. from Research Genetics (www.resgen.com ) </li></ul><ul><li>Control DNA from most strains is available e. g. from the Jackson Laboratory (www.jax.org) </li></ul>
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