Chimera 配種說明會

1. Chimera配種說明會 NRPGM基因轉殖鼠核心設施

2. Chimera配種說明會 • 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

3. 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 prevent unplanned matings • Coat color can be distinguished starting at about 8-9 days of age

4. 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

5. 配種方式 (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)。

6. 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.

7. 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.

8. http://www.jax.org/ http://140.112.133.74/ (使用者園地)

9. Chimera

10. ICR C57BL/6J       Chimera  C57BL/6J    R1 ES cell (129X1/SvJ x 129S1/Sv)F1

11. 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

12. 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

13. 配種紀錄(族譜)

14. Sizing Mouse Colonies 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 litters • Average number of pups per litter, average sex ratio per litter, and percent survival to weaning and adulthood • Breeder replacement schedules • Cage requirements, mouse room space, pair or trio breeding schemes, and allowable mouse density per cage (ACUC regulations)

15. Sizing Mouse Colonies

16. Sizing Mouse Colonies

17. AN EXAMPLE OF LOW-LEVEL MAINTENANCE 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.

18. 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.

19. The oldest male progeny can be discarded (shaded). No other action is necessary. Result: cage number reduced to five.

20. Nomenclature Guidelines for the Laboratory Mice

21. Rules for Nomenclature of Mouse and Rat StrainsRevised : July 2007 http://www.informatics.jax.org/mgihome/nomen/strains.shtml Rules for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations in Mouse and RatRevised: Jan, 2007 http://www.informatics.jax.org/mgihome/nomen/gene.shtml#genenom

23. Categories of characterized animals • Inbred strains • Substrains • Hybrids • F1 Hybrids • Recombinant Inbred Strains • Congenic Inbred Strains • Mixed Inbred Strains • Coisogenic Inbred Strains • Genetically Engineered strains • Transgenic • Targeted mutations (“knockouts” or “knockins”) • Consomic Strains, Segregating Inbred Strains & Conplastic Strains • Outbred Stocks

24. Inbred Strains/近親品系 • 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 • 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 • 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

25. 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 origin NZW (New Zealand White) • based on phenotype NOD (Non-Obese Diabetic) • others BALB/c, C57BL, 129

26. Standard Abbreviations

27. Substrains/支系 • 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

28. Nomenclature of Inbred Substrains [原始品系] / [序號(可省略)][實驗室編碼(Lab Code)]

29. A Laboratory Code • The Institute for Laboratory Animal Research (ILAR) maintains the International Laboratory Code Registry (Lab Codes). • The purpose of this database is to accurately identify at which laboratory or institution a strain of animals was bred and maintained. • Environmental factors can affect the characteristics of a strain, it is important to 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：National Applied Research Laboratories (國家實驗研究院 國家實驗動物中心) C57BL/6JNarl

30. Nomenclature of Inbred Substrains(continued) 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/6JOlaHsdEi C57BL = parent strain designation 6JOlaHsdEi = substrain designation 6 = line number J = laboratory code Ola = laboratory code Hsd= laboratory code Ei = Laboratory code

31. Congenic Inbred Strains/同源近親品系 • 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

32. A congenic strain is formed by backcrossing a locus of interest onto an inbred mouse strain for 10 or more generations.

33. Congenic inbred strain

34. Nomenclature of Congenic Inbred Strains [Recipient Strain*].[Donor Strain*]▬ [基因] *英文大寫縮寫 • 所導入之基因及其對偶基因來自於donor strain • B6.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

35. Nomenclature of Congenic Inbred Strains B6.Cg-Apoa1tm1Unc/J R1 ES cell (129X1/SvJ x 129S1/Sv)

36. 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 Nomenclature for Targeted Mutations 品系(/旁系)▬基因 tm[serial number][Lab Code] • 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 B6.Cg-Apoa1tm1Unc/J

37. 小鼠編號方式 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.

38. Figure 1.2. Dorsal view of a mouse showing commonly used numerical code approach for ear notching.

39. Figure 1.3. Ventral view of a mouse to orient feet. Digits can be amputated on neonatal mice as a form of numbering individuals.

40. 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.

41. nlac Mouse Genome Informatics (MGI) http://www.informatics.jax.org/ http://www.ncbi.nlm.nih.gov/genome/guide/mouse/ 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/

42. I-Shing Yu/游益興 Manager C-J Wa 王政儒 (2008) Animal room & DNA preparation Blastocyst injection & breeding of chimera ES cell targeting Construct production Southern blotting Z-S Huang 黃子珊(2006) C-P Wu 吳秋萍(2007) L-P Tzeng 曾莉萍(2007) H-Y Lin 凌凰翌 (since 2005, NTU’s core Y-P Yen 顏雅萍 (since 2005) C-L Hong 洪嘉倫 (2007) B-Y Wu 吳伯約 (2007)

43. 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