Data Report SeminarData Report Seminar

Ⅰ.Mice Feces ELISAⅡ.Gene ConstructionⅢ.TH1/TH2 TypingⅣ.Other Experiments

N509N509Tsai Ming-HANTsai Ming-HAN

Tsing Hua universityTsing Hua university 2002/12/62002/12/6

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Where did I work in this Where did I work in this summer?summer?

• IMB N509, Academic Sinica, Taipei.

• PI ： Dr. Nan-Shih Liao 廖南詩博士

What are N509’s What are N509’s works?works?

• Immunology.

• IL-15Rα Ko mice.

• The relationship of IL-15 and intestine.

Dr. Dr. 廖南詩博士廖南詩博士• 中央研究院分生所廖南詩老師主要是研究關於免疫方面，而目前主要的

方向是放在於 IL-15 及 IL-15Rα 與 T 細胞的關係。研究方向之一是利用IL-15Rα 基因刪除小鼠，研究 IL-15Rα 在 T 細胞發育及活化上的角色。我們發現 IL-15Rα-/- 小鼠缺少 natural killer 細胞， natural killer T 細胞， CD8αα+ 小腸表皮細胞間隙 T 細胞，及 CD8 T 細胞。其 CD8 T 細胞中 Bcl-2 表現量較低。 IL-15Rα-1- 小鼠對日本腦炎病毒感染造成之死亡亦較敏感。目前正在研究缺乏 IL-15Rα 造成這些異常表型的機轉。

• 另一方向是研究 IL-15 如何保護小腸表皮細胞間隙 T 細胞免於活化後的死亡。而實驗室希望找出負責這類 T 細胞活化致死的分子，也找出 IL-15Ｒ所傳遞之保護細胞存活的訊息。這些研究將幫助我們了解 T 細胞發育及活化的過程，以及 IL-15 及 IL-15Rα 在這些過程中的角色。

• 而我暑假研究的重心是放在第二項的研究上，也就是研究 IL-15Rα 基因刪除小鼠在小腸的免疫現象。所要負責測量的物質有IgA 、 IgM 、 IgG ，樣本是從老鼠的糞便中取得。

IL-15IL-15

• IL-15Rα-/- 小鼠缺少 natural killer 細胞， natural killer T 細胞， CD8αα+ 小腸表皮細胞間隙 T 細胞，及 CD8 T 細胞。其 CD8 T 細胞中 Bcl-2 表現量較低。 IL-15Rα-1- 小鼠對日本腦炎病毒感染造成之死亡亦較敏感

• 另一方向是研究 IL-15 如何保護小腸表皮細胞間隙 T 細胞免於活化後的死亡。我們希望找出負責這類 T 細胞活化致死的分子，也找出 IL-15 Ｒ所傳遞之保護細胞存活的訊息。這些研究將幫助我們了解 T 細胞發育及活化過程

MiceMice

• Mammal• Nice experiment animal • Gene similar to human

What data did I collect What data did I collect from mice? from mice?

• Source ： IL-15Ra wt 、 (he) 、 ko mice

• Sample ： Feces

• Targets ： IgA 、 IgG 、 IgM

• Method ： ELISA

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Mice Feces IgA IgG IgMMice Feces IgA IgG IgM

Collect feces and dry, add PBS and proteinase inhibtor 4C,o/n and coat plate, 4C, o/n.

Blocking plate 1hr, RT. At this time, dilute sample, after 1hr, Add sample and seal plate, place in 4C, o/n.

Add 2nd antibody, 1hr, RT. Then add avidin peroxidase, 30mins ,RT. Then add ABTS, read OD after 30 mins.

Collect data and discuss them by graph to find something.

DAY1DAY1

DAY2DAY2

DAY3DAY3

AFTERAFTER

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Read OD Data in Read OD Data in BiolinxBiolinx

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How to choose?How to choose?

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1.Count everyone standards if they has the best “twice” each other, and write down this “twice” OD range, as acceptable OD range.

2.Choose sample data as this OD range, give up those which data is far away from others.

3.Count the average of acceptable data, draw the graphs.

Standard1 1.2 1.8 0.69 1.93 0.36 1.92 0.19 1.95 0.1 2.2 0.04Standard2 1 1.7 0.66 1.89 0.35 2.1 0.17 2.1 0.08 2.6 0.03Standard3 1.1 1.8 0.63 1.89 0.34 2.2 0.15 2.08 0.07 1.97 0.04

Why chose this data?Why chose this data?

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Mice Feces IgA Mice Feces IgA Separate by AgeSeparate by Age

IgA of Mice IL-15KO feces

0

100

200

300

400

500

600

700

ng/m

l

F

M

M

F

F

M

M

M

M

M

F

F

M

M

M

F

M

IgA of mice WT feces

0

50

100

150

200

250

300

350

ng/m

g

F

F

M

M

M

M

F

F

F

M

M

1 1.5 2 3.5 1 1.5 2 month month

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IgA of mice IL-15Ra HE feces

0

50

100

150

200

250

300ng

/mg

F

M

M

F

M

Month 1.5 3.5

Mice Feces IgM Mice Feces IgM Separate by AgeSeparate by Age

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IgM of mice IL-15Ra Ko feces

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

ng/m

g

F

M

M

F

F

M

M

M

F

F

M

M

M

F

M

IgM of mice WT feces

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

ng/mg

F

F

M

M

M

F

F

F

M

M 1 1.5 2 3.5 1 1.5 2monthmonth

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IgG of mice IL-15Ra KO feces

0

1

2

3

4

5

6

ng/mg

FMM

FFMMMMM

FFMMM

FM

IgG of mice WT feces

0

1

2

3

4

5

6

7

8

9

10

ng/mg

F

F

M

M

M

M

F

F

F

M

M

Mice Feces IgGMice Feces IgG Separate by Separate by AgeAge

1 1.5 2 3.5 1 1.5 2monthmonth

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IgG of mice IL-15Ra HE feces

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

ng/m

g

F

M

M

F

M

Month 1.5 3.5

IgA of mice feces

0

100

200

300

400

500

600

700

0 0.5 1 1.5 2 2.5 3 3.5 4

months

ng/m

g

KOWTHE

IgM of mice feces

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.5 1 1.5 2 2.5 3 3.5 4

month

ng/m

g

KO

WT

IgG of mice feces

0

1

2

3

4

5

6

7

8

9

10

0 0.5 1 1.5 2 2.5 3 3.5 4

month

ng/m

g

KO

WT

HE

Serum IgA.M.G ELISASerum IgA.M.G ELISA

0500

100015002000250030003500

5th 5th 8th 8th 8th 8th

4mon 4mon 2mon 2mon 2mon 2mon

WT KO WT HE WT KO

FA2 FA1 FA1 FB2 FB4 FB1

21202 21202 50102 50102 50102 50102

61702 61702 62502 62502 70102 70102

#1 #2 #3 #4 #5 #6

IgA(μ g/ml)

IgM(μ g/ml)

IgG(μ g/ml)

ConclusionConclusion

• In mice feces, IgA>> IgG> IgM .

• In mice serum, IgG>> IgM> IgA .

• In mice feces, IgA has low conc at 1.5mons.

• We can briefly find that female has more immunoglobins than male.

• Antibody concentration maybe has a great relationship to age and sex, but it depends on large data.

• Needs to collect more data to prove what we suggest.

QUESTION & PROBLEMQUESTION & PROBLEM

• We find that IgA IgM OD is too high, and its read is much higher than standard, so next time I will dilute sample IgA and IgM more.

• We can’t use half-well plate or we need to change conc of standards and samples.

• When wash by PBST, it’s easily to get some water on plate, we can use a plate to cover it.

FUTURE WORKFUTURE WORK

• Collect more data to make a conclusion.

• Collect different situation mice feces.

(Feed with bacteria)

Gene ConstructionGene Construction

PGL2B

PGL2B 0.4+0.7(In exp, all gel use 0.8%)

What did I do?What did I do?

• 學長從一段 DNA 序列中挑出一段可能為promoter 的序列，並將他 clone 到 pGL2B 的plasmid 上之後，也做了 luciferase 確定他是promoter 沒錯。而我所要做的事情，是將這段序列的 down stream 序列切掉一些，來看看這個 promoter 的表現，是否有 operater 存在。不過這個實驗沒做完。而 pGL2B 質體是一種去除 promoter 的質體，其 MCS 之後有一段螢光蛋白的基因，可藉由不同的 promoter 來表現它而進而推知這個 promotet 的強度。

PGL2B PGL2B

PGL2B 0.4+0.7PGL2B 0.4+0.7

PGL2B 0.4+0.7 Insert PGL2B 0.4+0.7 Insert GeneGene

If YaYa success….If YaYa success….PGL2B-1032SAPGL2B-1032SA

EXP 2-1EXP 2-1

Use Sac1 to check if the bacteria are my needs.

1kb PG1 PG2 PG471 PG472

H2O 13.8

DNA 2

BSA 10x

2

Sac1 0.2

NEB1 10x

2

20

Choose PG2 and PG472 and use restriction enzyme, 4hrs or more.

PG*2

DNA 15

Buffer(4) 10x

5

Sma1 3

H2O 27

50

PG47*2

DNA 15

Buffer(4) 10x

5

BSA 10x 5

Sac1 2

Apa1 3

H2O 20

50

1kb PG2 PG472

5.5kb 1.2kb

5.5kb

1kb

200bp

EXP 2-1 0verEXP 2-1 0ver

Vector gel purify (PGL2B*2)

Insert gel purify (PGL2B47*2)

vector

insert

vector

EXP 2-1 DiscussionEXP 2-1 Discussion

• Before gel purify, never use short wavelight UV light, it may induce mutate.

• When gel purify, use 50V to get more clear DNA bends.

EXP 3EXP 3

1kb PG1 PG2 471 472

H2O 13.8

DNA 2

BSA 10x

2

Sac1 0.2

NEB1 10x

2

20

H2O 6.9

DNA 1

BSA 10x

1

Sac1 0.1

NEB1 10x

1

10

Use Sac1 check find that 4 tubes are all PGL2B 0.4+0.7

EXP 3EXP 3

1kb PG47*1 PG47*1 PG*1 PG*2

H2O 6.9

DNA 1

BSA 10x

1

Sac1 0.1

NEB1 10x

1

10

H2O 0

DNA 60

BSA 10x

6

Sac1 3.2

NEB1 10x

6

75

Use Sac1 to cut PG47*1 Use Sac1 to check PG*1 PG*2 and double check if PG47 cut over or not.

Supercoild

1.2kb

Supercoild

1.2kb

EXP 3EXP 3

1kb 100bp PG2 47*1 1kb 100bp PG2 47*1

1.2kb

1.2kb

Supercoild

Supercoild

PG*2

DNA 35

Buffer(4) 10x

7

Sma1 5

H2O 23

70

Cut PG*2 by Sma1, and check if PG47 cut over or not

Double check if PG and PG47 cut over or not

EXP 3EXP 3

Cut PG47*2 because PG47*1 didn’t cut well, and triple check PG*2

1kp PG*2 PG47*1

H2O 43

DNA 30

BSA 10x

10

Sac1 7

NEB1 10x

10

100

PG*2 ok, use kit purify plasmid. Then CIP it .

H2O 139

DNA 40

CIP 1

NEB4 10x

20

200

0,7264x3.5/40x0.6=0.38

0.38*40*3.03/6=7.6U

What’s new?What’s new?

• CIP (Alkaline Phosphate, Calf Intestinal)

(Prevent from self-ligation)

• Mung Bean Nuclease

(Cut off 3’ and 5’ extensions from DNA or RNA)

Blunt and extension endsBlunt and extension ends

• Without extension affinity

• Will have a problem about 2 directions ligation.

So, blunt ends are more difficult to ligate.

(~><~)

Other experimentOther experiment

• Typing

• IgG subtypes titration

• Work in the animal room

• Watching what others do

GenotypingGenotyping

• Cut 2mm mice tail.• Use Hot lysing method. (50mM NaOH, 60min

incubation, then heat at 95 degree, 30min).• Use Tris to neutralize. Centrifuge.• Use supernatant for PCR.

IgG subtypes titrationIgG subtypes titration

• Titration for the best amount of 1st and 2st ab for substrate.

• To determine the best and economical concentration for us to use in the exp.

Lab MembersLab Members

THANKS^^THANKS^^