Genetical Standardisation and Quality Control PDF

Genetical standardisation and quality control Genetical standardisation what is necessary for the experiment? – uniformity (transplant-experiments, cancer research, …) – variation (testing medicins, toxicology, …)  Genetical standardisation reproducibility is...

Genetical standardisation and quality control Genetical standardisation what is necessary for the experiment? – uniformity (transplant-experiments, cancer research, …) – variation (testing medicins, toxicology, …)  Genetical standardisation reproducibility is important genetic background should be standardised can be achieved … by using specific breeding techniques (inbred and/or selection), genetical variation in a population can be manipulated biotechnical procedures (in-vitro fertilisation, transgenesis, cloning) increase the speed to reach the wanted breeding results Genetical standardisation major factor contributing to the variation of the results in animal experiments = genetic background of the animals  Effect of background each strain has unique background alleles → may interact with and modify expression of mutation, transgene, or other genetic insert in uncharacterized background or mixed background of unspecified origin → bigger chance of modifier genes having confounding effect modifier genes are reason why normal development and physiology often vary significantly among inbred strains. Effect of background  Effect of background diabetes (db) and obese (ob) mutations ((Coleman and Hummel 1973; Coleman 1978) – B6 background: obesity and transient diabetes – C57BLKS/J (BKS) background: obesity and overt diabetes Alzheimer's amyloid precursor protein transgene, Tg(APP695) ( (Carlson et al. 1997) – outbred mice: merely induces the formation of amyloid plaques in the brain – inbred FVB/N and B6 mice: lethal IL10-deficiency (Beckwith et al. 2005) – B6 background: slightly increases susceptibility to inflammatory bowel disease (IBD) – 129/SvEv, BALB/c, and C3H/HeJBir backgrounds: greatly increases IBD susceptibility Effect of environment  Genetical uniformity monozygote animals inbred strains F1-hybrid co-isogenic lines congenic lines chromosome-substitute strain recombinant inbred strains recombinant congenic lines Monozygote animals in nature: genetic variation still limited amount of genetical uniformity Î nine- banded armadillo (Dasypus novemcinctus), per nest: – 1 fertilised egg cell – 4 monozygote pups → genetically identical genetical identical ≠ complete phenotypical identity or identical response to procedures subtle differences during intra-uterine development → small changes in phenotype → variation is smaller than in animals with different genetical backgrounds  Monozygote animals if genetic uniformity is required, monozygotic individuals are the animals of choice. – only a limited number of animals are available produce monozygote animals by cloning produce monozygote animals by inbreeding Monozygote animals beginning 20th century: – cancer research demands genetically uniform animals – tumour tissues maintained by successive transplantation – not always succesfull because of rejection – production of inbred strains gets started at the moment > 400 mouse and rat-inbred strains  Inbred strains inbred = breeding closely related individuals resulting in an increase in homozygosity of the pups degree of inbreeding → expressed as inbred coefficient F = fraction of original heterozygous genes that become homozygous Inbred strains the more generations of inbreeding, the more F increases increase of F per generation is determined by the relation between the parents (degree of cosanguinity) Inbred: after at least 20 successive generations of brother x sister-breeding or mating of offspring x youngest parent inbred coëfficiënt F will be 98.4% = on average 98.4 % of the originally heterozygous loci have become fixed in a homozygous state still some variation, but quite genetically uniform = isogenic strain Inbred coefficient  Inbred strains in inbred strains growth, fertility and vitality can decrease – most properties are set by different genes (polygenic) – in natural populations: combinations of genes that function best and that survive the natural selection, are the most common = balanced system Inbred strains in any natural population – number of detrimental recessive alleles – not expressed in the phenotype – each one accompanied by dominant counterpart random mating: the chance is small of a descendant becoming homozygous for a recessive allele mating between related parents: this chance increases  Inbred strains – by inbreeding individual is fixed into an arbitrary combination of genes not necessarily the most beneficial → inbred depression animals function worse compared to the original population effects of unfavourable alleles are more pronounced in homozygous condition Inbred strains – first 4-8 generations: lot of drop outs because of unfavourable gene combinations – > 10 generations: most unfavourable alleles dropped out new balanced system develops  Inbred strains - indication capital letters – DBA, WAG – some old strains also have numbers (C3H, C57Bl6) substrains – spontaneous mutations and/or environmental influences (changes in the genome) – in nomenclature: code behind the slash, e.g. C57Bl6/J, C3H/Ola Substrains  Inbred strains - characteristiques Approved abbreviations for common mouse strains AK AKR strains B C57BL B6 C57BL/6 strains B10 C57BL/10 strains C BALB/c strains C3 C3H strains CB CBA D1 DBA/1 strains D2 DBA/2 strains J SJL SW SWR  Inbred strains genetic defects compared to human diseases, e.g. obesity, muscular dystrophy, hydrocephalus (at the left normal black mouse, mouse at the right has rounded, enlarged skull, typically for hydrocephalus) Inbred strains Co-isogenic strain spontaneous mutations in inbred strain → differentiates just in 1 gene of original strain = co-isogenic strain substrain can be maintained next to original inbred strain, e.g. when mutant represents genetic model for a human disease or involves a gene of general interest pro’s: research in one particular gene control group is available  Inbred strains Co-isogenic strain nomenclature: full strain and substrain designation, followed by dash and indication of mutant gene e.g. C57BL/6J-Aqp2cph/J (congenital progressive hydronephrosis ) not always easy to maintain certain substrains, depending on mutated gene some only maintainable in heterozygous state (backcrossing) Inbred strains Co-isogenic strain Mouse gene – italics, first letter capitalized Adenomatosis polyposis coli = Apc Leptin receptor = Lepr Mouse allele – italics, superscipted First letter capitalized if dominant – ApcMin First letter lower case if recessive - Leprdb  Inbred strains F1-hybrid F1 hybrids (Filial 1) resulting from a cross between two inbred strains features of F1 hybrids that make them particularly useful include the following: – genetically and phenotypically uniform – possess hybrid vigor more resistant to disease survive better under stress live longer have larger litters than either parental strain → heterosis Inbred strains F1-hybrid features of F1 hybrids that make them particularly useful include the following: – useful as hosts for tissue transplants (e.g., tumors, skin, and ovaries) from either parental strain. – for many studies, they are more viable than are the parental strains.  Inbred strains F1-hybrid in some cases F1 hybrid react more uniform than the strains they originate from, e.g. reacting more uniformly to pentobarbital under certain circumstances F1 hybrid can show more variation than the parents → due to the improved ability of the F1 to adapt (= Tryon effect) → perform pilot experiments Inbred strains F1-hybrid F1 hybrids generally more uniform respons: morphological parameter more variable respons: behavioural characteristics can make a difference which strain gives the mother or the father animals  Inbred strains F1-hybrid both the NZW and NZB mice exhibit an elevated fasting blood sugar level when compared to Swiss white mice NZB/NZW F1 hybrid mouse shows still a higher fasting serum glucose level than either of its parental strains (B. Pansky et al., 1975) Inbred strains F1-hybrid nomenclature: uppercase abbreviations of the two parents (maternal strain listed first), followed by F1 reciprocal F1 hybrids are not genetically identical Î designations are different – D2B6F1 mouse Î offspring of a DBA/2 mother and C57BL6/J father − full F1 designation is (DBA/2N x C57BL/6J)F1 – B6D2F1 mouse Î offspring of the reciprocal cross − full F1 designation is (C57BL/6J x DBA/2N)F1  Inbred strains Congenic strain introducing certain genetic qualities into an inbred strain by backcrossing repeatedly (e.g. nude gene) most used method to bring in recessive characteristic: cross-intercross-backcross method recessive characteristic mm of strain D (donor) is imported in strain A after initial cross, a cycle of intercrossing- backcrossing is repeated at least 10 times (animals selected for the backcross must carry the gene of interest mm) Inbred strains Congenic strain as result of crossing-over during meiosis and selection of proper backcross animals, donor gene is introduced into the genome of strain A → practically identical strain, except for the gene that was selected and some genes of donor strain D to the left and right of the mutant gene, that will be reduced with successive generations of backcrossing  Congenic strain Inbred strains Congenic strain symbol → three parts: full or abbreviated symbol of recipient strain separated by a period from abbreviated symbol of donor strain hyphen separates strain name from symbol (in italics) of differential allele(s) introgressed from the donor strain (in cases where chromosome on which the mutation arose is unknown, e.g., donor is not inbred or is complex or an F1 hybrid, the symbol Cg should be used to denote congenic)  Inbred strains Congenic strain Examples B6.AKR-H2kA mouse strain with the genetic background of C57BL/6 but which differs from that strain by the introduction of a differential allele (H2k) derived from strain AKR/J. LEW.BN-RT1nA rat strain with the genetic background of LEW but which differs from that strain by the introduction of a differential segment (RT1n) derived from strain BN. DA.F344-Cia5A rat strain with the genetic background of DA onto which a segment from the F344 strain containing the Cia5 QTL has been transferred. B6.Cg- KitW-44J Gpi1aA mouse strain with the genetic background of C57BL/6, but where the donor strain is mixed, the Kit allele originating from C3H/HeJ and the Gpi1 allele originating from CAST/Ei. Inbred strains Congenic strain by using DNA-markers: speed congenic method (just need 5-7 back-crossings) double congenic lines are produced to study interactions between genes  Inbred strains Chromosome-substitution strain inbred strain where chromosome is replaced by homologeous chromosome of another inbred strain (donor) by repeated backcrossings (minimum of 10 generations) = chromosome-substitution-strain = consomic strain complete panel of strains, – each originating from same donor and same acceptor – each with different chromosome replaced by chromosome of the donor Inbred strains Chromosome-substitution strain nomenclature for consomic strains is HOST STRAIN-Chr #DONOR STRAIN name of host strain is not abbreviated strain of origin is shown in superscript capitalization of all letters in superscript and non-italicization of the chromosome letter/number and of the superscript distinguish a chromosome identifier from an allele symbol.  Inbred strains Chromosome-substitution strain Examples – SHR-Chr YBN Y chromosome from BN has been backcrossed onto SHR C56BL/6J-Chr 19SPR M.spretus Chromosome 19 has been backcrossed onto C57BL/6J C56BL/6J-Chr 1A/J Chr 3DBA/2J Chromosome 1 from A/J strain and Chromosome 3 from DBA/2J strain have been backcrossed onto C57BL/6J Inbred strains Chromosome-substitution strain phenotypical analysis of a complete panel allows fast association of a phenotypic quality with a certain chromosome range of congenic strains can be made, where chromosome is divided into segments → position of a certain locus is being refined (by backcrossing and identification of the pups)  Inbred strains Chromosome-substitution strain Chromosomes 6 and 13 Harbor Genes that Regulate Pubertal Timing in Mouse Chromosome Substitution Strains (Krewson et al, Endocrinology vol 145, 2004) evaluated pubertal timing [assessed by vaginal opening (VO)] in A/J and C57BL/6J (B6) + in a panel of chromosome substitution strains (CSSs) generated from A/J and B6 mice VO o significantly earlier in A/J compared with B6 mice o majority of CSSs had timing of VO similar to progenitor B6 strain o CSSs for chromosomes 6 and 13 each displayed significantly earlier time of VO than B6 mice. chromosomes 6 and 13 harbor Quantitative Trait Loci that control the timing of VO. identification of responsible genes may reveal factors that regulate maturation of Hypothalamic-Pituitary-Gonadal axis and determine timing of puberty. Inbred strains Recombinant inbred strain recombinant inbred strains are produced by mating individuals from F2 generation of a cross between 2 inbred strains (also called progenitor strains) → after minimum 20 generations brother x sister = RI-strain (recombinant-inbred strain) → each RI-strain represents a fixed set of randomly assorted genes of both progenitors  Inbred strains Recombinant inbred strain nomenclature: combining abbreviated names of both parental strains which are separated by a capital “X” parallel lines are given numbers, e.g. B6XH-1 is the first recombinant inbred strain derived from progenitor strains C57Bl/6 and C3H/HeJ  Inbred strains Recombinant inbred strain recombinant inbred strains are very valuable for genetic research studies on linkage analysis (genes that are inherited together) identification and genetic analysis of complex genetic traits, since linked loci are more likely to be transmitted together Inbred strains Recombinant congenic strain recombinant congenic strains (RCS) represent a series of inbred strains, which are derived from the second or third backcross generation of 2 unrelated progenitor strains, one serving as background and one as donor (e.g. base strain carrying tumours)  Inbred strains Recombinant congenic strain after at least 20 generations of inbreeding, genome of each of the resulting recombinant congenic inbred strains will primarily contain genomic material from the background strain and a small proportion of the donor genome after 2 backcrosses on average 12.5% of the donor genome is unevenly dispersed over the background strain various chromosomal segments overlap partly → 20-25 parallel lines are needed to represent at least 95% of the genome of the donor strain  Inbred strains Recombinant congenic strain nomenclature: prefix RCS and the abbreviated names of the progenitor strains separated by a small “c” e.g. RCS CcS-1 is the first recombinant congenic strain from the parental strains balb/c (background) and STS (donor) (STS is susceptable to chemically induced colon tumors) used to study genetic background of multifactor characteristics (susceptibility to tumour development, disease resistance) Random-bred population genetic uniformity not always needed, e.g. for research in the field of how medicines work, a heterogenic group is more interesting not easy to keep such a strain genetically constant → in a closed population there is always a certain amount of inbreeding → the smaller the population, the bigger the chance of inbreeding  Random-bred population If matings happen ad random, increase of the inbred coefficient per generation is ∆ F = 1/(8nf) + 1/(8nm), where nf and nm is the number of male and female breeders If nf ≅ nm then ∆ F = 1/(2N), where N is the total number of breeding animals Suppose 50 females and 50 males, then ∆ F = 0,5% Suppose 10 females and 10 males, then ∆ F = 2.5% Inbred coefficient  Random-bred population by increasing the inbred coefficient the allele pool of a population decreases per generation rotation schemes keep the relationship between breeding animals as small as possible Poiley rotation scheme (1960) The population is divided into 12 groups, each with a minimum of 12 monogamous breeding pairs. In each generation the following mating scheme between groups is applied: Breeding Groups Female Male (Sire) Offspring (Dam) 12 10 1 1 11 2 2 12 3 3 1 4 4 2 5 5 3 6 6 4 7 7 5 8 8 6 9 9 7 10 10 8 11 11 9 12 e.g.: a Group #5 female is mated with a Group #3 male. Their offspring then belong to Group #6.  Outbred-strain if a random-bred-population is being kept as a closed colony during at least 4 generations and the increase of the inbred coefficient (F) does not exceed 1% per generation = outbred-strain at least 25 breeding pairs ∆ F = 1/8.25 + 1/8.25 = 1/200 + 1/200 = 2/200 = 1/100 = 0.01 = 1% Outbred-strain designation of an outbred strain symbol indicating the current breeder of the stock colon stock symbol consisting of between 1 and 4 capital letters Mouse: HsdWin:NMRI Crl:CD-1 Rat: HsdBrlHan: Wistar Hsd:Sprague Dawley Crl:LE (Long Evans)  Outbred-strain phenotypic variation is greater in outbred strains than in inbred strains – study of sleeping time under hexobarbital anesthesia mean sleeping time in 5 inbred strains ranged from 18 to 48 minutes with a SD of 3.2 minutes averaged across the strains mean sleeping time in 2 outbred strains of 43 and 48 minutes with a standard deviation of 13.5 minutes → same range, but much higher SD → can obscure the effects of treatment → need much more animals to get reliable data Outbred-strain Hybrid population or mosaic population during long term experiments, in particular if consistent genetic variation is essential, use of an outbred stock is inadequate due to gradual change in gene pool. genetic variation can be standardised through a system of reciprocal hybridising of inbred strains for long-lasting experiments, where a certain variation of the population is wanted, same genetic variance can be made over and over again  Outbred-strain Hybrid population or mosaic population with 4 inbred strains A, B, C and D at least 10 different genotypes can be produced. Î with n = number of inbred strains, n+(n²-n)/2 genotypes can be produced Guidelines for Nomenclature of Mouse and Rat Strains http://www.informatics.jax.org/nomen/strains.shtml  Genetical quality control inbred strains must be preserved very accurately → years of work can get lost if strains get mixed! genetic contamination is not always phenotypically clear (e.g. mixing of 2 albino strains) genetical quality control is necessary keeping the uniformity of inbred strains controlled Genetical quality control uniformity of inbred strains is tested by skin transplants: → accepting or rejecting the transplant is dependent of number of histocompatibility loci that are located on different chromosomes → rejection if donor and receptor are no longer identical  Genetical quality control uniformity of inbred strains is tested by describing number of genetical characteristics (if possible monogenic) of the strain: → colour genes, immunogenetic markers, biochemical genetical markers → variants of enzymes (isozymes) and other proteins, present in blood and tissue, are used (gel-electrophoresis and afterwards specific colouring → zymogram Zymogram  Genetical quality control uniformity of inbred strains is tested by use of DNA-markers: − markers based on variation (polymorphy) in the nucleotides sequence of the DNA − 2 types of DNA-markers − type I: polymorphism in expressing gene, the so-called functional genes, from which the function is known Genetical quality control 2 types of DNA-markers type II: polymorphism in DNA of which the function is not (yet) known (most used) SSLP (simple sequence length polymorphism): variation in difference in length of microsatellites (short pieces of DNA where a sequence of 2-5 nucleotides is repeated 10-30 times (tandem-repeats, eg. CACACACACA). These microsatellites are very polymorph and differences in length are the consequence of differences in number of repeats of the base unit via PCR: amplification of microsatellites, then electrophoresis and making visible → visualising length differences  Cryopreservation freezing of embryos/sperm purpose: keeping unique strains of becoming extinct (infection, accident) avoiding ‘genetic drift’ preserving valuable mutants avoiding unnecessary breeding (decreasing number of used animals) sometimes easier to transport than living animals Cryopreservation after breeding: collecting embryos (8-cell stage, early morula, 3 days after CP) → freezing (‘vitrification’) embryo is put in liquid that draws out all the water → temperature is decreased very fast → kept in liquid nitrogen problem: formation of crystals after defreezing → implant into receptive female (pseudo-pregnant after mating a vasectomized male) ICSI: intracytoplasmatic sperm-injection in oocyt