genetic monitoring of laboratory animalsnm.kalas.or.kr/eml/2018_ltdata/2018_02_yoon.pdf ·...
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Genetic Monitoring of Laboratory Animals
Korea Research Institute of Bioscience and BiotechnologyLaboratory Animal Resource Center
Won Kee Yoon, D.V.M., PhD, DKCLAM
유전적배경차이가표현형에미치는영향
유전모니터링마커
형태학적마커 (Morphological Markers)
생화학적마커 (Biochemical Markers)
면역학적마커 (Immunogenetical Markers)
분자유전학적마커 (Molecular Genetic Markers)
형태학적마커모니터링
Coat color 관련유전자 C & c; pigmented와 non-pigmented를결정(Tyrosinase: Tyr)
• C/C, C/c: 유색• c/c : 다른유전자에우선해서 albino(백색피모, 적색눈)
• cch : chinchilla
A & a; agouti와 non-agouti를결정• A/A, A/a: 색소가털의선단및모근부에는밀도있게,
중앙부에는엷게분포• a/a : 전부위에고르게밀도있게분포• Aw : white-bellied agouti
B & b; black과 brown을결정• B/B, B/b : 흑색색소를합성• b/b : 갈색색소를합성• A/-, B/- : 흑색색소의불균일한분포에의해서agouti (야생쥐색)
• a/a, B/- : 흑색색소의균일한분포에의해서 black
형태학적마커모니터링
Coat color 관련유전자 D & d; non-dilute와 dilute를결정(Myosin Va: Myo5a)
• d/d : 색소과립이응집되어분포하므로모색이엷어짐 p ; pink-eyed dilution
ln ; leaden
Pink-eyed, light-bellied chinchilla leaden
형태학적마커모니터링
Coat color
Strain ColorChromosome 2 4 7 9 -
Locus a b c d *
A/HeAKRBALB/cCBA/JC3H/He
WhiteWhiteWhiteBlack-agoutiBlack-agouti
aaAAA
bBbBB
cccCC
DDDDD
C57BL/6C57BL/10C57BR/cdC57LC58
BlackBlackBrownLeadenBlack
aaaaa
BBbbB
CCCCC
DDDDD
ln(1)
DBA/1DBA/2DDKKKNC
Dilute brownDilute brownWhiteWhiteBrown-agouti
aaAaA
bbBBB
CCccC
ddDDD
NZBNZWRIIISJL129
BlackWhiteWhiteWhiteLight chinchilla
aAAAAw
BbBBB
Cccccch
DDDDD
p(7)p(7)
형태학적마커모니터링
Coat color test mating
P P F1
DBA/2aabbCCddDilute brown
AKRaaBBccDDalbino
aaBcCcDdblack
HRSAabbccddalbino
AabbCcddyellow
BALB/cAAbbccDDalbino
AabbCcDdBrown-agouti
C57BL/6aaBBCCDDBlack
BALB/cAAbbccDDalbino
AaBbCcDDBlack agouti
A/a; agouti/non-agouti, B/b; black/brownC/c; color/albino, D/d; non-dilute/dilute
생화학적마커모니터링
대부분의생화학적마커는 isoenzymes
Locus symbol Gene name Chromosome
No. Alleles Tissues Supporting media
Ihh1 Isocitrate dehydrogenase 1 1 a, b kidney CA
Pep3 Peptidase 3 1 a, b, c kidney CA
Akp1 Alkaline phosphatase 1 1 a, b kidney CA
Car2 Carbonic anhydrase 2 3 a, b RBC CA
Mup1 Major urinary protein 1 4 a, b Urine PAGE
G6pd1 Glucose-6-phosphate dehydrogenase 4 a, b Kidney CA
Pgm1 Phosphoglucomutase 1 5 a, b Kidney CA
Ldr1 Lactate dehydrogenase regulator 1 6 a, b RBC CA
Gpi1 Glucose phosphate isomerase 1 7 a, b Plasma CA
Hbb Hemoglobin beta-chain 7 s, d, p RBC CA
Es1 Esterase 1 8 a, b Plasma CA
Es2 Esterase 2 8 a, b Kidney CA
Mod1 Malic enzyme 1 9 a, b Kidney CA
Trf Transferrin 9 a, b Plasma CA
Es3 Esterase 3 11 a, b, c Kidney CA
Mouse Biochemical Markers Monitored at KRIBB (15항목)
생화학적마커모니터링
대부분의생화학적마커는 isoenzymes
Locus symbol Gene name Chromosome No. Alleles Tissues Supporting
media
Hbb Hemoglobin beta chain 1 a, b RBC CA
Amy1 Amylase 1 2 a, b Pancreas CA
Svp1 Seminal vesicle protein 1 3 a, b Seminal fluid CA
Mup1 Major urinary protein 1 5 a, b Urine CA
Es6 Esterase 6 8 a, b Testis PAGE
Akp1 Alkaline phosphatase 9 a, b Kidney CA
Alp1 Serum alkaline phosphatase 1 9 a, b Serum CA
Gc Group-specific components 14 a, b Serum PAGE
Es1 Esterase 1 19 a, b Intestine PAGE
Es2 Esterase 2 19 a, c, d Plasma PAGE
Es3 Esterase 3 19 a, b, c, d Intestine PAGE
Es4 Esterase 4 19 a, b Kidney CA
Es7 Esterase 7 19 a, b Testis PAGE
Es8 Esterase 8 19 a, b Testis PAGE
Es9 Esterase 9 19 a, c Testis PAGE
Es10 Esterase 10 19 a, b Testis CA
Es14 Esterase 14 19 a, b, c Serum PAGE
Rat Biochemical Markers Monitored at KRIBB(17항목)
면역학적마커모니터링
대상마커종류
Thy1 (Thymus cell antigen 1), Chr. 9, spleen (thymus)
H-2K (Histocompatibility-2K), Chr.17, spleen (lymph node)
H-2D (Histocompatibility-2D), Chr.17, spleen (lymph node)
실험방법
FACS analysis
Thy1; Thy1.1(CD90.1), Thy1.2(CD90.2)
H-2K; H-2Kb, H-2Kd, H-2Kk, H-2Kq
H-2D; H-2Db, H-2Dd, H-2Dk, H-2Dq
면역학적마커모니터링
Alloantigen Chart
Strain Name Thy1 H2K H2D
129/J 2 b b
C57BL/6J 2 b b
BALB/cA 2 d d
C3H/HeJ 2 k k
CBA/J 2 k k
DBA/1J 2 q q
DBA/2J 2 d d
NOD 2 d b
분자유전학적마커모니터링
사용되는마커종류
RFLP (restriction fragment-length polymorphisms)
SSLP (simple sequence length polymorphism) or Microsatellite
markers
SNP (single nucleotide polymorphism)
분자유전학적마커모니터링
SNPs (Single Nucleotide Polymorphisms)
Jackson Lab SNP ID Chr No. SNP ID Alleles
AF067836_350A_1 1 rs3022796 A/T
M22381_169_2 1 rs3022825 C/T
M-09011_1 2 rs3022883 A/C
M-02707_1 3 rs3022953 C/T
M-01609_1 4 rs3022977 C/T
M-02187_2 5 rs3023034 G/T
M-05233_3 5 rs3023039 A/G
M-02094_1 6 rs3023064 G/T
M-05782_1 7 rs3023106 G/T
M-08396_1 7 rs3089205 C/G
M-11559_2 8 rs3023177 A/C
M-05537_1 9 rs3023203 A/G
M-04819_1 9 rs3023226 C/T
M-09526_2* 10 rs3089984 G/T
M-05799_1 10 rs3089109 C/G
M-08924_1 11 rs3088673 A/C
M-05727_1 11 rs3023251 C/T
M-07403_3 12 rs3023342 C/T
M68896_151_1 13 rs3023381 C/T
M-05495_2 13 rs3023382 G/T
M-07251_1 14 rs3091048 G/T
M-07646_1 15 rs3091174 A/G
M-01322_2 16 rs3023436 A/T
AF027865_1 17 rs3023450 C/T
M-04659_1 17 rs3023442 A/G
M-09844_1 18 rs3089349 A/G
M-02162_1 19 rs3023481 A/G
M-05810_1 X rs3089604 C/T
꼬리에서 DNA추출
SNP Marker를이용하여 Real Time PCR실시
SNP Marker를이용한 Inbred mouse strain(96 strain)
PCR 결과분석
Petkov et al. Genomics 83 (2004)
분자유전학적마커모니터링
SNPs (Single Nucleotide Polymorphisms)
꼬리에서 DNA추출
SNP Marker를이용하여 Real Time PCR실시
SNP Marker를이용한 B6J와 B6N strain 구분
PCR 결과분석
SNP ID Chromosome No Alleles
rs13477019 3 T/A
rs13478783 6 A/G
rs13479522 7 A/G
rs13480122 9 T/C
rs13480619 10 T/C
rs29359333 10 G/A
rs13480759 10 C/T
rs13481014 11 T/C
rs13481734 13 A/G
rs31233932 14 G/A
rs4165065 16 T/C
rs13483055 17 T/C
Mekada et al. Exp Anim 58 (2009)
유전학적품질유지
믿을수있는 breeding source로부터동물도입.
Use proper nomenclature to describe your
mouse models.
Include a detailed description of the genetic
background of the mice you use in all your
communications.
정기적인유전모니터링실시
냉동보존
Cryopreservation
Advantages
It saves space.
It saves money.
It insures against catastrophic loss (fire, flood, earthquakes,
disease, etc.).
It provides a method for eliminating pathogens from a
mouse colony.
It reduces the risk of a strain contamination.
It dramatically slows the rate of genetic drift.
It reduces the risk that transgene copy number will change.