genetic monitoring of laboratory animalsnm.kalas.or.kr/eml/2018_ltdata/2018_02_yoon.pdf ·...

Genetic Monitoring of Laboratory Animals

Korea Research Institute of Bioscience and BiotechnologyLaboratory Animal Resource Center

Won Kee Yoon, D.V.M., PhD, DKCLAM

목 적

비의도적인교배에따른유전학적오염, genetic

drift에의한유전학적변이여부를찾아내기위함

☞본래그계통이가지고있던특성을그대로

유지하여올바른실험결과획득

유전적배경차이가표현형에미치는영향

http://www.wellesley.edu/Chemistry/Chem101/diet/hughs/leptin.jpg

http://www.wellesley.edu/Chemistry/Chem101/diet/hughs/leptin.jpg

유전모니터링대상형질(마커)의조건

유전적으로발현이명확할것

안정적으로발현되는것

결과가명확하여판정이용이한것

소량의시료로도가능한것

경제적일것

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


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


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)


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)


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



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)


실험기자재

Applicator Well plate

Aligning base Titan III membraneElectrophoresis system


염색및결과판정

Idh1 Akp1 Car2


염색및결과판정

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


결과판독 – Thy1


결과판독 – H-2K


결과판독 – H-2D


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

생화학 & 면역학적마커모니터링

분자유전학적마커모니터링

사용되는마커종류

RFLP (restriction fragment-length polymorphisms)

SSLP (simple sequence length polymorphism) or Microsatellite

markers

SNP (single nucleotide polymorphism)


SSLP or Microsatellite

Schalkwyk et al. Genome Res 879, 1999


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)



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)



C57BL/6J → C57BL/6N

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.

genetic monitoring of laboratory animalsnm.kalas.or.kr/eml/2018_ltdata/2018_02_yoon.pdf ·...

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