Embed Size (px)
Citation preview
Chimera 配種說明會
NRPGM 基因轉殖鼠核心設施
• 小鼠配種方式簡介• 基因剔除鼠配種說明• 基因剔除鼠命名• 小鼠編號方式
Chimera 配種說明會
Some important “normal” mouse breeding characteristics
• Sexual maturity at roughly 5-8 weeks of age• Estrus cycle: 4-5 days• 19-21 day gestation period• Average litter size: 6-8 pups• Pups must be weaned at 3-4 weeks of age to preve
nt unplanned matings • Coat color can be distinguished starting at about 8-9 days of age
Some important “normal” mouse breeding characteristics
• Female fertility starts to decline at about 6-8 months of age, while males usually
remain fertile for at least a year • Females can become pregnant within a
day of giving birth (postpartum estrus) • Multiple females can be mated with a
single male, but not vice versa • Males used for breeding must not be
housed with other males
(1) 持續交配：雌鼠被允許永久與雄鼠及群體其他成員相 處並產下仔鼠。此系統乃利用產後發情方式繁殖。a . 一夫一妻：一隻公鼠一隻母鼠關在一起。b . 一夫多妻：一隻公鼠及多隻母鼠關在一起。
(2) 暫留交配；在分娩之前將公鼠和母鼠暫時分開。a . harem 系統，公鼠及母鼠關在一起至分娩前才分開。b. 人工控制交配系統；公鼠和母鼠關在一起一段充分的時間進行交配 (通常約 1個星期 )。
配種方式
PHEROMONE EFFECTS
• Lee-Boot EffectHousing female mice in groups will result in synchronization of their estrus cycles. Prolonged absence of male pheromones results in a state of anestrus (lack of a normal estrus cycle).
• Whitten EffectEstrus can be induced in most group-housed females by adding male mouse urine (or dirty bedding from a male’s cage) to their cage.
• Bruce EffectPheromones from a strange male can prevent the implantation of embryos into the uterine walls of a recently bred female. This is why one should not move a female from one male’s cage to another.
Replacement of breeders
Replace a breeder pair if:
• they have not produced a litter in more than two months,
• they are producing small litters (1-3 pups per litter),
• they are killing their pups.
Chimera
R1 ES cell(129X1/SvJ x 129S1/Sv)F1
Chimera
C57BL/6J
C57BL/6J
ICR
From Chimera to Homozygous Null (129 ES cells injected into C57BL/6 host blastocysts)
• Chimeric mouse ♂ x C57BL/6 ♀ = B6129F1 and/or C57BL/6
• B6129F1 coat color agouti, 50% carry targeted alleles +/-
• B6129F1 ♂ +/- x B6129F1 ♀ +/- = B6129F2
• B6129F2 coat color mixed, 25% homozygous -/- founders
• B6129F2 ♂ -/- x B6129F2 ♀ -/- = B6129F3
• B6129F3 coat color mixed, 100% homozygous -/-
• B6129F3 are used for initially characterizing targeted genes
From 129 to C57BL/6 Congenic
• B6129F1 ♂ +/- x C57BL/6 ♀ +/+ = B6129N1 25% +/-
• B6129N1 ♂ +/- x C57BL/6 ♀ +/+ = B6129N2 25% +/-
• B6129N2 ♂ +/- x C57BL/6 ♀ +/+ = B6129N3 25% +/-
• B6129N3 ♂ +/- x C57BL/6 ♀ +/+ = B6129N4 25% +/-
• B6129N4 ♂ +/- x C57BL/6 ♀ +/+ = B6129N5 25% +/-
• .
• .
• B6.129N10 ♂ +/- x B6.129N10 ♀ +/- = B6.129N10F1 25% -/-
• B6.129N10F1 ♂ -/- x B6.129N10F1 ♀ -/- = B6.129N10F2 100% -/-
• B6.129N10F2 -/- congenics are suitable for study on defined genetic background
配種紀錄 (族譜 )
To properly size a research mouse colony, many factors must be considered, including the following:
• Number of mice needed, utility of each sex, needs for specific genotypes and age-matched mice
• Number of strains needed (for example, a cre-lox experiment may require three breeding colonies)
• Preferred breeding scheme• Strain productivity, genotypes and phenotypes affecting productivity,
and number of unproductive matings• Female’s reproductive life span, average number and frequency of li
tters• Average number of pups per litter, average sex ratio per litter, and p
ercent survival to weaning and adulthood• Breeder replacement schedules• Cage requirements, mouse room space, pair or trio breeding schem
es, and allowable mouse density per cage (ACUC regulations)
Sizing Mouse Colonies
Sizing Mouse Colonies
Sizing Mouse Colonies
Mat.cage 1
Mat.cage 2
Holding cage
Holding cage
Holding cage
Holding cage
7 pups 5 pups 4 males5 females
3 males 4 males
3 wks old
2 wks old
15 wks old
15 wks old
10 wks old 8 wks old
The progeny from mating cage 1 are ready to be weaned. Three females and four males are found. They are numbered and placed in two cages. The two cages of older progeny are discarded (shaded boxes). Result: no change in cage number.
AN EXAMPLE OF LOW-LEVEL MAINTENANCE
Mat.cage 1
Mat.cage 2
Holding cage
Holding cage
Holding cage
Holding cage
8 pups 5 pups 3 males 4 males 4 males 3 females
newborn
3 wks old
11 wks old 9 wks old 4 wks old 4 wks old
The progeny from mating cage 2 are ready to be weaned. Two females and three males are found and numbered. The females are added to the cage of 3 females created in week 1, while the males are put into a new cage. The oldest cage of male progeny is discarded (shaded). Result: no change in cage number.
Mat.cage 1
Mat.cage 2
Holding cage
Holding cage
Holding cage
Holding cage
8 pupsNo pups
4 males 4 males 5 females 3 males
1 wk old
9 wks old 5 wks old4&5 wks old
4 wks old
The oldest male progeny can be discarded (shaded). No other action is necessary. Result: cage number reduced to five.
Nomenclature Guidelines for the Laboratory Mice
Rules for Nomenclature of Mouse Rules for Nomenclature of Mouse and Rat Strainsand Rat Strains
Revised : July 2007
http://www.informatics.jax.org/mgihome/nomen/strains.shtml
Rules for Nomenclature of Genes, GeRules for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations netic Markers, Alleles, and Mutations in Mouse and Ratin Mouse and RatRevised: Jan, 2007 http://www.informatics.jax.org/mgihome/nomen/gene.shtml#genenom
nomenclature
gene protein
mouse
基因符號以斜體英文字母，第一個字母大寫，第二個以後小寫
Apc, adenomatosis polyposis coli
蛋白質符號全部大寫，不需斜體
APC
human
基因符號以斜體英文字母，所有字母大寫
APOA1, apolipoprotein A1
蛋白質符號全部大寫，不需斜體
APOA1
Categories of characterized Categories of characterized animalsanimals
Inbred strainsInbred strains Substrains
HybridsHybrids F1 Hybrids
Recombinant Inbred StrainsRecombinant Inbred Strains Congenic Inbred StrainsCongenic Inbred Strains
Mixed Inbred Strains Coisogenic Inbred StrainsCoisogenic Inbred Strains Genetically Engineered strainsGenetically Engineered strains
Transgenic Targeted mutations (“knockouts” or “knockins
”) Consomic Strains, Segregating Inbred StraiConsomic Strains, Segregating Inbred Strai
ns & Conplastic Strainsns & Conplastic Strains Outbred StocksOutbred Stocks
Inbred StrainsInbred Strains// 近親品系近親品系 定義定義
兄妹近親交配 (brother x sister mating) 20 代或 20 代以上，且在此品系之 20 代或接續的世代中，每一個個體皆可追溯自單一個來源之祖先對。
優點優點 遺傳性狀及表現性狀具一致性 (only 0.01 residual heterozygosity) 品系特殊性 性狀穩定 , 國際流通性佳… ..etc.
缺點缺點 近親抑制現象 (inbreeding depression) 如體型小、低繁殖率、生命期短等 Strain-specific characteristics 昂貴
Nomenclature of Inbred Strains
具簡潔獨特性之符號，以英文大寫字母或字母連結數字表示based on coat color
DBA (Dilute Brown nonAgouti, first inbred mouse)
based on originNZW (New Zealand White)
based on phenotypeNOD (Non-Obese Diabetic)
othersBALB/c, C57BL, 129
Standard AbbreviationsStandard AbbreviationsAbbreviation Strain Abbreviation Strain
129P 129P3/J L C57L/J
129S 129S1/SvImJ CBACa CBA/CaGnLe
AHe A/HeJ CB CBA
A A/J C3 C3H/HeJ
AK AKR/J C3Fe C3HeB/FeJ
CBy BALB/cByJ D1 DBA/1J
C BALB/cJ D2 DBA/2J
B C57BL NZB NZB/BlNJ
B6 C57BL/6J NZW NZW/LacJ
B10 C57BL/10J SJL or J SJL/J
BR C57BR/cdJ SW SWR/J
SubstrainsSubstrains// 支系支系 定義定義
近親品系之繁殖族群與其共同祖先分離超過 20 個世代以上，或族群之維持完全獨立於其原始品系之外而形成支系
Nomenclature of Nomenclature of Inbred SubstrainsInbred Substrains
[ 原始品系 ] / [ 序號 ( 可省略 )][ 實驗室編碼 (Lab Code)]
A Laboratory CodeA Laboratory Code The Institute for Laboratory Animal Research (ILAR) maintains the Internation
al Laboratory Code Registry (Lab Codes). The purpose of this database is to accurately identify at which laboratory or in
stitution a strain of animals was bred and maintained. Environmental factors can affect the characteristics of a strain, it is important t
o document where a strain originated
http://dels.nas.edu/ilar_n/ilarhome/labcode.shtml
J ： The Jackson Laboratory N ： National Institutes of Health Crl ： Charles River Laboratories Rik ： The Institute of Physical and Chemical Research (RIKEN) Narl Narl ：： National Applied Research LaboratoriesNational Applied Research Laboratories
( 國家實驗研究院 國家實驗動物中心 )
C57BL/6JNarl
由於遺傳的變異隨時間的演進而出現，故實驗室編碼 (Lab Code) 應隨品系的轉移而累加於後
C57BL = parent strain designation 6JOlaHsdEi = substrain designation 6 = line number J = laboratory code Ola = laboratory code Hsd = laboratory code Ei = Laboratory code
Nomenclature of Inbred SubstrNomenclature of Inbred Substrainsains
(continued)
C57BL/6JOlaHsdEi
Congenic Inbred Strains/Congenic Inbred Strains/ 同源同源近親品系近親品系
定義定義 某外源基因和連帶的一段染色體由他品系 (donor strain) 以
雜交方式導入受領品系 (recipient strain; background strain) 內，經回交 (backcross) 或回交 / 互交 (backcross-intercross) 10 代以上，並經適當的遺傳標記篩選所育成之新品系
同源近親品系除了在同一基因區之某幾對基因與原來之近親品系有所不同，保持分離外，其在遺傳上之特性與原品系幾乎完全相同
5≦N＜ 10 generations are considered incipient congenic
A congenic strain is formed by backcrossing a locus of interest onto an inbred mouse strain for 10 or more generations.
Congenic inbred strainCongenic inbred strain
Nomenclature of Nomenclature of Congenic Inbred Congenic Inbred StrainsStrains
所導入之基因及其對偶基因來自於 donor strainB6.AKR-H2k
recipient strain ： C57BL/6J donor strain ： AKR
B6.129P2-Tcrbtm1Mom
recipient strain ： C57BL/6J donor strain ： 129P2 via E14TG2a ES cell line
若 donor strain 不是 inbred ，而是雜交、遺傳背景混雜或未知時，以“ Cg ” 表示之
B6.Cg-Tcrbtm1Mom
[Recipient Strain*].[Donor Strain*] ▬ [ 基因 ]
*英文大寫縮寫
Nomenclature of Nomenclature of Congenic Inbred StrCongenic Inbred Strainsains
R1 ES cell (129X1/SvJ x 129S1/Sv) B6.Cg-Apoa1tm1Unc/J
Nomenclature for Nomenclature for Targeted Targeted MutationsMutations
以 tm 代表 targeted mutation Serial number : 來自於實驗室原始編號 Lab Code :突變株所來自之實驗室
129X1-Cftrtm1Unc
the first targeted mutation of the cystic fibrosis transmembrane regulator (Cftr) gene at 129X1, produced at the University of North Carolina
knock-out
129X1-En1tm1(Otx2)Wrst
the coding region of En1 was replaced by the Otx2 gene, originating from the W. Wurst laboratory
knock-in
品系 (/旁系 )▬ 基因 tm[serial number][Lab Code]
B6.Cg-Apoa1tm1Unc/J
Figure 1.1. A small metal ear punch can be used to mark the ears of mice at specific locations to create a numerical code for individual mouse identification.
小鼠編號方式
Figure 1.2. Dorsal view of a mouse showing commonly used numerical code approach for ear notching.
Figure 1.3. Ventral view of a mouse to orient feet. Digits can be amputated on neonatal mice as a form of numbering individuals.
Figure 1.4. Tattoo ink can be injected into the skin at specific locations, as indicated, in neonatal mouse feet to create a numerical code to identify individuals.
Mouse Genome Informatics (MGI) http://www.informatics.jax.org/
http://www.ncbi.nlm.nih.gov/genome/guide/mouse/
nlac
國家實驗研究院 國家實驗動物中心http://www.nlac.org.tw/
實驗動物資訊網http://las.nhri.org.tw/
L-P Tzeng曾莉萍 (200
7)
C-L Hong洪嘉倫 (2
007)
Z-S Huang黃子珊 (2006)
H-Y Lin凌凰翌(since 2005, NTU’s core
Y-P Yen顏雅萍
(since 2005)
B-Y Wu吳伯約 (2007)
C-P Wu吳秋萍(2007)
Blastocyst injection & breeding
of chimera
ES cell targeting
Southern blotting
Construct productio
n
Animal room &
DNA preparatio
n
I-Shing Yu/ 游益興Manager
C-J Wa王政儒 (2008)
說明• 本投影片檔關於命名的部分 Slide係參考吳希天及周傳凱博士的上課講義
