藉由豬胰澱粉酶啟動⼦基因轉殖策略賦予轉基因豬同時表現外源植酸分解酶與纖維素分解酶之可行性

國⽴立臺灣⼤大學⽣生物資源暨農學院動物科學技術學系 博⼠士論⽂文

Department of Animal Science and Technology College of Bioresources and Agriculture

National Taiwan University Doctoral Thesis

藉由豬胰澱粉酶啟動⼦子基因轉殖策略賦予轉基因豬同時 表現外源植酸分解酶與纖維素分解酶之可⾏行性 The Feasibility of a Transgenic Strategy for Co-expression of Phytase and Cellulase Transgenes Driven by the Porcine

Pancreatic Amylase Promoter in Pigs

林育聖 Yu-Sheng Lin 指導教授：鄭登貴 博⼠士 徐濟泰 博⼠士

Advisors ：Winston Teng-Kuei Cheng, Ph.D Jih-Tay Hsu, Ph.D.

中華民國 103 年 10 ⽉月

October 2014

 

 

誌 謝

本論⽂文能順利完成並付梓，乃承蒙恩師 鄭登貴教授在研究、︑､課業及⽣生活上給予 極⼤大之指導、︑､關懷與⿎鼓勵， 徐濟泰教授不辭⾟辛勞悉⼼心提供諸多寶貴建議， ⿈黃⽊木秋教 授、︑､ 陳全⽊木教授、︑､與 吳信志教授在學位考試及論⽂文上之詳細審閱斧正；⽼老師們勉勵 提攜之情，讓學⽣生在博⼠士班訓練過程中獲益良多並感念於⼼心，謹此致最深之謝忱。︒｡

在研究過程中，同時⾮非常感謝 徐啟真博⼠士、︑､與 楊卓真博⼠士於實驗細節上之提點 以及論⽂文的校審；朱有⽥田教授在實驗儀器上的⽀支援；劉嚞睿教授、︑､余碧教授提供酵素 活性測試⽅方法指導；農場鄭位明學⾧長、︑､重威、︑､及阿芬阿姨在樣本採樣以及試驗動物照 顧之協助；劉秀洲博⼠士、︑､廖奕雯學姐、︑､游位育學⾧長於平⽇日⽣生活上的激勵和學務上的協 助；以及動物⽣生殖科技研究室志任、︑､淳淳、︑､華雲、︑､劭于、︑､妍樺、︑､廉本、︑､碩甫、︑､家駿、︑､

冠宇、︑､偉誠，營養研究室瑋蕾、︑､柏元、︑､煜軒，分⼦子⽣生物研究室虹妏、︑､晁偉、︑､原佑等昔

⽇日與今⽇日的實驗室成員在精神與試驗上的⿎鼓勵與協助，能與各位共同學習以及切磋分 享，實是⼈人⽣生中最珍貴的回憶。︒｡

最後要感謝我的家⼈人，謝謝你們在這些年來給予的⽀支持，遂使求學⽣生涯能無後顧 之憂，得以順利完成學業。︒｡

謹以本論⽂文獻給我敬愛的家⼈人，及所有關⼼心我的朋友，謝謝你們！

謹識於

國⽴立台灣⼤大學 動物科學技術學系 中華民國 103 年 10 ⽉月 22 ⽇日

 

中⽂文摘要

藉由豬胰澱粉酶啟動⼦子基因轉殖策略賦予轉基因豬同時表現外源 植酸分解酶與纖維素分解酶之可⾏行性

林育聖

近年全球⾯面臨氣候變遷與能源轉型、︑､短缺，造成國際榖物供需失衡，國內進⼝口

⼤大⾖豆、︑､⽟玉⽶米等主要飼料原料價格⾼高漲，養豬產業飼料成本節節上揚，且畜體飼養過程 中所產⽣生之排泄物、︑､廢棄物與其所衍⽣生之環境污染更是⽬目前養豬業者務必嚴正⾯面對之 環保課題；職是之故，部分研究先進乃嘗試從營養設計之⾓角度，試圖改良動物對營養 分之消化吸收，從⽽而降低⼤大量營養物質隨糞便排出之⽐比例，進⽽而得能有效減少飼料浪 費與環境污染之⽬目標。︒｡

本研究旨在釐清吾等先前已成功產製以豬胰澱粉酶基因啟動⼦子在胰臟中表現 外源之植酸分解酶與纖維素分解酶之轉基因豬，是否具備有效提⾼高消化率與降低糞磷 之排放能⼒力；試驗之進⾏行包括：1. 完成分析業經成功選殖2,488-bp之豬胰澱粉酶基因 啟動⼦子序列特性以及其活性；2. 依據業經建⽴立之轉基因豬，完成分析並確認彼等外 源植酸分解酶與纖維素分解酶等之轉殖基因，分別在其體內之組織表現性及其在性腺 被傳承之能⼒力；3. 進⾏行表⾯面消化率試驗，裨謀⽐比較彼等⾮非轉基因豬與轉基因豬，分 別被飼予對照組⽇日糧（control diet）與/或低磷⽇日糧（low-P diet）時，各受試豬隻對於 飼糧中包括乾物質、︑､粗蛋⽩白質、︑､粗纖維、︑､鈣、︑､磷等之利⽤用的差異性。︒｡

試驗結果證明：1. 前述業經選殖出之2,488-bp序列⽚片段被與綠⾊色螢光蛋⽩白基因 共同構築成報導載體後，確實能在⼤大⿏鼠胰臟腫瘤細胞株（AR-42J cell line）中成功表 現出螢光蛋⽩白質，證明該選殖⽚片段確定具備啟動⼦子之活性，且能有效控制下游基因之 表現；2. 以聚合酶反應（polymerase chain reaction, PCR）及南⽅方吸漬法（Southern blot）

檢測F1世代基因組，證明先前試驗所產製之轉基因豬具備性腺傳承之能⼒力。︒｡此外藉由 反轉錄聚合酶鏈鎖反應（reverse transcription-PCR）策略分析F1世代之器官及⁄或組織，

結果證實包括植酸分解酶與纖維素分解酶兩者之mRNA表現，分別皆具備有組織之專

⼀一性，且僅在胰臟組織中被轉錄。︒｡進⼀一步藉由西⽅方吸漬（Western blot）分析包括F1 與F2世代轉基因豬之胰臟組織及其腸內容物；結果證明彼等源⾃自被轉殖之植酸分解酶 與纖維素分解酶等外源基因，其所表現之蛋⽩白質分別確能在胰臟被合成，且經由胰管 分泌進⼊入⼗十⼆二指腸中。︒｡此外，經活性試驗結果證明，前述兩種蛋⽩白質在胰臟及⼗十⼆二指 腸內容物，且分別具備分解植酸與纖維素之活性，並以⼗十⼆二指腸內容物活性較⾼高；植 酸分解酶活性經過鉬藍法（molybdate-blue method）定量計算結果，證明其在⼗十⼆二指 腸中存在，且⾼高達到8.2 U/mL之譜；3. 在以⽟玉⽶米、︑､⼤大⾖豆及⼤大⿆麥為主之對照組⽇日糧中，

相較於彼等⾮非轉基因豬⽽而⾔言，轉基因豬在乾物質、︑､中性洗滌纖維、︑､粗蛋⽩白質、︑､鈣、︑､及 磷等之表⾯面消化率，分別均有較佳之表現，特別是乾物質及中性洗滌纖維等，更呈現 有顯著性之差異（P <0.05）；就飼予⽟玉⽶米、︑､⼤大⾖豆及⼤大⿆麥等為主之低磷組⽇日糧者⽽而⾔言，

轉基因豬在乾物質、︑､中性洗滌纖維、︑､粗蛋⽩白質、︑､鈣、︑､及磷等之表⾯面消化率，分別均較 彼等⾮非基因轉殖豬者，有較佳的表現（P <0.05）。︒｡其屬有趣者，乃⾮非基因轉殖豬⽐比較 之被飼予對照組及/或低磷組⽇日糧者，並進⼀一步分析其糞便中之氮及磷含量者，試驗

結果顯⽰示轉基因豬之被餵飼對照組與低磷組⽇日糧者，其糞便中磷之排出量分別被減少

29%和48%之譜，此外其糞便中氮排出量亦分別下降多達31%和49%之譜。︒｡

綜合上述試驗結果，本研究證明，試驗所構築之2,488-bp豬胰澱粉酶基因啟動

⼦子序列不僅具有啟動轉錄之能⼒力，且在先前產製之轉基因豬所攜帶之外源基因，確定 可經由性腺被傳承⾄至其⼦子代。︒｡此外，彼等在胰臟組織專⼀一表現植酸分解酶及纖維素分 解酶之轉殖基因，其表現之外源轉基因之蛋⽩白質，且分別具備有分解酶之活性，遂能 有效增進其對飼料之消化效率從⽽而顯著減少糞中氮及磷之排出量。︒｡未來之試驗研究冀 望得能進⼀一步完成豬隻在各不同⽣生⾧長階段之相關營養試驗，並針對此等轉基因豬隻各 階段之⽣生⾧長速率，對於各不同飼料原料之消化能⼒力，及豬隻在腸道中被免疫相關基因 之調控等，可能分別扮演有重要之⾓角⾊色；深盼透過本轉基因殖豬模式之完成建⽴立，不 僅得能賦予豬隻具備有效利⽤用⾼高纖維原料替代物（fiber-rich by products）之固有營養 分，且能有效維持豬隻⽣生⾧長與繁殖性能，從⽽而更能兼具臻於環保⽬目標之要求。︒｡

關鍵字: 轉基因豬, 豬胰澱粉酶基因啟動⼦子, 植酸分解酶, 纖維素分解酶

Abstract

The Feasibility of a Transgenic Strategy for Co-expression of Phytase and Cellulase Transgenes Driven by the Porcine Pancreatic Amylase Promoter

in Pigs

Lin, Yu-Sheng

Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in both corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of by-products and alternative ingredients in pig diets has the potential to reduce feed costs. However, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down non-starch polysaccharides into simple sugars.

In addition, pigs do not produce sufficient endogenous phytase to hydrolyze P from phytate in cereals, making it necessary to supplement with inorganic phosphorus (P) to meet the requirements for optimum pig production, resulting in higher fecal concentrations of P and increasing potential environmental pollution. Thus, increased regulatory pressures are being imposed on animal agriculture to minimize P and nitrogen emissions. Improvement of the digestive efficiency of pigs is the most logical approach for reducing nutrient excretion and achieving pollution control. This study investigated a biological method for reducing fecal P excretion and improving nutrient digestibility, and utilized a transgenic strategy in which

expression of bacterial phytase and fungal cellulase in the gastrointestinal tract to establish an eco-friendly pig model.

A 2,488-bp 5′-flanking region of the porcine pancreatic amylase gene was cloned and its structural features were characterized. Using green fluorescent protein as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Germ-line transmission and tissue-specific expression of phytase and cellulase transgenes in the pancreas of F1 and F2 transgenic pigs were identified. Both enzyme activities were also detectable in pancreas and duodenal contents. Up to 8.233 U/mL phytase activity was detected in the duodenal contents of transgenic pigs, which represents 5−8 times the activity in non-transgenic pigs. On a corn, barley, and soybean-meal-based control diet, the apparent fecal digestibility of dry matter (DM), neutral detergent fiber (NDF), crude protein, calcium, and P was higher in transgenic pigs than in non-transgenic pigs (P < 0.05 for both DM and NDF digestibility). On a low-P basal diet, the transgenic pigs exhibited significantly increased digestibility of these nutrients (P < 0.05).

Conclusions come to the above studies indicated that those transgenic pigs fed with the control and/or the low-P diet(s) would result in significantly decrease of their fecal P outputs by 29% and 48%, respectively, and also their fecal N outputs was reduced by 31% and 49%, respectively, when comparisons were made to the fecal P and/or N outputs from those non-transgenic pigs. Based on evidences demonstrated above, the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene and suggests our transgenic pigs may represent a way to increase nutrient utilization and reduce fecal P and N contents.

Keywords

: transgenic pig, pancreatic amylase promoter, phytase, cellulase

Table of Contents

Page

⼝口試委員審定書

... I

誌謝

... II

中⽂文摘要

... III

Abstract

... VI

List of Figures

... XIV

List of Tables

... XVII

Introduction

... 1

Chapter 1 Review of the Literature

... 4

1.1 The Digestive Tract of Pigs... 4

1.1.1 Mouth and Salivary Glands... 4

1.1.2 Stomach... 6

1.1.3 Small Intestine... 7

1.1.4 Large Intestine... 8

1.2 Pancreatic Secretions in the Pig... 9

Page

1.2.1 Structure of the Pancreas………...…………. 9

1.2.2 Enzyme Secretion of the Exocrine Pancreas…...……... 10

1.3 Exogenous Enzymes in Porcine Feeding Programs………... 15

1.3.1 Carbohydrase Supplementation………..…... 17

1.3.2 Phytase Supplementation………..……… 21

1.3.3 Enzyme Combinations………..…… 27

Chapter 2 Characterization of a Putative Pancreatic Amylase Gene Promoter From a Pig

... 28

2.1 Objective... 28

2.2 Materials and Methods... 29

2.2.1 Computer-based Analysis: the 5'-flanking Region of the Porcine Pancreatic Amylase Gene... 29

2.2.2 Cloning of the Fluorescent Protein Reporter Construct... 30

2.2.3 Culture of Rat Pancreatic AR-42J Cells…... 30

Page

2.2.4 Transfection and Transient Expression of the AMY-GFP

Reporter Construct... 31

2.3 Results... 31

2.3.1 Characterization of the Putative Porcine Pancreatic Amylase Gene Promoter Region... 32

2.3.2 In Vitro Activity of the Porcine Pancreatic Amylase Gene Promoter... 32

2.4 Discussion... 33

2.5 Summary... 36

Chapter 3 Evaluation of Transgenic Pigs Expressing AMY-PHY and AMY-CEL in the Gastrointestinal Tract

... 39

3.1 Objective... 39

3.2 Materials and Methods... 40

3.2.1 PCR and Southern Blot Analyses………..……. 40

Page

3.2.2 RNA Extraction and Analyses of RT-PCR………. 41

3.2.3 Protein Extraction... 42

3.2.4 Cloning and Purification of Recombinant Proteins... 43

3.2.5 Western Blotting Analyses... 45

3.2.6 Phytase Enzyme Extraction……... 46

3.2.7 Assessment of the Phytase Enzyme Activity... 46

3.2.8 Assessment of the Cellulase Enzyme Activity... 48

3.2.9 Statistical Analyses……...…... 49

3.3 Results... 49

3.3.1 Germ-line Transmission in Transgenic Pigs……….…….. 49

3.3.2 Transgene Expression in the Pancreas... 50

3.3.3 Phytase Activity Analyses for Transgenic Pigs... 51

3.3.4 Cellulase Activity Analyses for Transgenic Pigs... 51

3.4 Discussion... 52

Page

3.5 Summary…... 54

Chapter 4 Potential of the Transgenic Pig for Improvement of Their Digestive Efficiency

…... 64

4.1 Objective…... 64

4.2 Materials and Methods…... 65

4.2.1 Phosphorus-digestibility Trials…... 65

4.2.2 Chemical Analyses of Diets, Feces, and Blood Serum…... 66

4.2.3 Statistical Analyses……...…... 67

4.3 Results…... 68

4.3.1 Chemical Analyses of Feces in Transgenic Pigs Expressing the AMY-CEL Transgene…... 68

4.3.2 Chemical Analyses of Feces in Transgenic Pigs Expressing the AMY-PHY Transgene…... 68 4.4 Discussion... 69

Page

4.4.1 Phytase Enzyme Activity in Transgenic Pigs…... 70

4.4.2 Cellulase Enzyme Activity in Transgenic Pigs... 72

4.4.3 Digestibility of Nutrients in Transgenic Piga……….……... 74

4.5 Summary... 76

General Conclusion and Future Works

... 82

References

... 84

Appendices

... 104

List of Figures

Page Figure 1

Diagrammatic representation of the porcine digestive system... 5

Figure 2

Cascade of biochemical events starting with proenteropeptidase action.……….. 13

Figure 3

Inefficiency associated with digestion in pigs.……….…….

16

Figure 4

The plant cell wall……….……...

18

Figure 5

The structure of phytate……….………

24

Figure 6

The 5'-flanking region of the porcine pancreatic amylase gene.………….……...

37

Figure 7

Verification of promoter activity……….………..

38

Figure 8

Identification of transgenic pigs harboring phytase and cellulase

transgenes.………..…….……….…….. 56

Page

Figure 9

RT-PCR analysis of phytase and cellulase expression in the F1 generation

transgenic pig.……….……... 57

Figure 10

Western blot analysis of phytase and cellulase expression in the pancreas of F1 and F2 generation transgenic pigs ………...………..……

58

Figure 11

Western blot analysis of phytase expression in the duodenal contents of an F2 generation transgenic pig.………..………

59

Figure 12

Time course of expression of cellualse enzyme in porcine tissues………….…...

60

Figure 13

Western blot analysis of cellulase expression in the duodenal contents of an F2 generation transgenic pig.………..

61

Figure 14

Apparent nutrient digestibility in nontransgenic and transgenic pigs………..…..

77

Figure 15

Impact of genetic modification on porcine fecal phosphorus (P) and nitrogen (N) outputs on a control diet.……….…………..……….….

78

Page Figure 16

Apparent nutrient digestibility in nontransgenic and transgenic pigs fed low-P diet………..

79

Figure 17

Impact of genetic modification on porcine fecal phosphorus (P) and nitrogen (N) outputs on a low-P diet.………..…..……….…………..

80

List of Tables

Page Table 1

Regulation of protein synthesis in rat exocrine pancreas.………. 12

Table 2

Mean concentration of non-starch polysaccharides in cereals and some legume

grains.………... 19

Table 3

Total and phytate phosphorus content in feed.…………..……… 22

Table 4

Common phytase enzymes used in swine nutrition.……….…………. 26

Table 5

Rate of germ-line transmission of F0 generation transgenic pigs expressing AMY-PHY and AMY-CEL…... 61

Table 6

Phytase activity in the pancreas and gastrointestinal tract lumen of F2

generation transgenic pigs.……….……… 62

Table 7

Composition of the experimental diets.……….……… 80

 

Introduction

The environmental issues surrounding pig production present a growing

public-health concern, particularly with regard to the disposal of feces. Feeds are

supplemented with minerals and inorganic phosphorous (P) to meet the nutritional

requirements of the porcine diet; excess intake is readily excreted in the feces and

results in water pollution and soil erosion (Carpenter, 2008). Although phase feeding is

effective for reducing P and nitrogen (N) excretion in porcine feces, the low

digestibility of P-bound phytate (phytate-P) is the primary concern (Dourmad and

Jondreville, 2007). The use of phytase, which releases phytate-bound minerals in feeds,

has gained popularity in the industry (Harper et al., 1997; Chu et al., 2009).

The corn and soybean meal in Taiwan are imported products. The fluctuation of

their prices becomes one of the most influencing factors for pig production (Saengwong

et al., 2011). In 2008, the prices of grain and soybean has increased, substantially

causing pig production cost per 100 kg live hog weight raised from NT$ 5,146 (in 2007)

to NT$ 6,566 (National Animal Industry Foundation, 2012). To overcome the global

increase in the cost of traditional feeds and limited cereal production, local and

inexpensive ingredients are to be used as substitutes for more expensive conventional

ingredients in porcine diets (Esteban et al., 2007; Weber et al., 2008; Taiwan Grain and

Feed Annual, 2014); however, most of these alternatives comprise fiber-rich

by-products and have distinct nutritional profiles in comparison to common traditional

ingredients (Varel and Yen 1997). Therefore, improved fiber conversion for energy

utilization will provide critical reductions in grain consumption and digestive waste in

the pig industry.

Feedstuffs of plant origin contain phytic acid within the cell, and non-starch

polysaccharides (NSPs) are located in the cell walls. Phytase and carbohydrates may act

in synergy to improve nutrient utilization, as the carbohydrases hydrolyze the NSP in

cell walls to increase phytase access to phytic acid (Woyengo and Nyachoti, 2011). In

the previous study (Lin, 2004), transgenic pigs harboring transgenes for bacterial

phytase and fungal cellulase were generated by using a direct co-injection strategy.

These animals have since been anticipated being capable of showing much better to

digest those fibers, use P more efficiently, and produce much less solid waste;

ultimately bringing much more profit to farmers. To allow spatial and temporal control

of transgene being expressed in this type of pig, a promoter DNA fragment was cloned

from the porcine pancreatic amylase gene subsequently being designed for driving both

of the exogenous phytase and cellulase genes. However, it remains to be determined

that if the cloned amylase promoter is sufficient to drive both of the phytase and

cellulase transgenes, and whether these secreted proteins can maintain their enzymatic

properties in the duodenal lumen. The objective of Experiment I was to characterize the

properties of this putative promoter as well as being able of providing fundamental

information regarding transgene expression in those transgenic pigs generated.

Experiment II was designed to verify the expression and activity of phytase and

cellulase in the pancreas and gastrointestinal tract. To verify the feasibility of producing

adequate digestive enzymes for efficient digestion in transgenic pigs carrying

transgenes driving by the porcine pancreatic amylase promoter, further studies related to

the digestive efficiency was measured in Experiment III. The valuable information

obtained from these studies should be conducted for providing those important clues

regarding to the utility of these transgenic lines of pigs as a promising alternative

strategy for ensuring though events related to hydrolysis of partial anti-nutritional

compounds in diets.

Chapter 1 Review of the Literature

1.1 The Digestive Tract of Pigs

Pigs have a monogastric or called as the non-ruminant digestion system. Similar

to all other monogastric animals, their digestive tract is highly developed and has five

main parts, including the mouth, esophagus, stomach, and small and large intestines

(Yen, 2001a; Figure 1).

1.1.1 Mouth and Salivary Glands

The digestive process starts with feed entering the mouth of the pig. The physical

action of chewing promotes salivary glands secretion by reflex nervous stimulation

(Strube, 2013). More than 90% of the saliva is derived from the secretion of three pairs

of major glands, the parotid, mandibular, and sublingual glands, which secrete proteins

belonging to the classes of proline-rich proteins, mucins, lysozyme, immunoglobulin (Ig)

A, and α -amylase (Nagler, 2008). The activity of porcine salivary α -amylase, which

hydrolyzes the α-1,4-glycosidic linkages of starch and oligosaccharide and is secreted

(Yen, 2001a)

Figure 1. Diagrammatic representation of the porcine digestive system.

by the sublingual glands, is low and largely inactivated by the acidic gastric contents

(Araki et al., 1990). From a quantitative viewpoint, salivary α-amylase is not considered

important because the ratio of total salivary amylase to total pancreatic amylase in the

postprandial phase (0

–

5 h after feeding) is only about 1:250,000 (Lærke and Hedemann, 2012). Thus, although α-amylase is secreted in the oral cavity, starch digestion is not of

quantitative importance here because the time spent by the feed in the mouth is too short

(Yen, 2001b).

1.1.2 Stomach

The pig stomach is a reservoir hold about 30% of the total volume of the

digestive tract (Wenk, 2001). Compared to ruminants, pigs have a “simple” stomach in

which only slight microbial modification of available nutrients takes place before

absorption occurs. Digestion of ingested proteins begins with pepsin activity within an

optimum pH range from 2 to 3 (Rajagopalan et al., 1966). When feed enters the

stomach, it encounters hydrochloric acid and pepsin, both of which may compromise

exogenous enzyme stability and activity via acid denaturation and proteolytic digestion

(Strube et al., 2013). However, pepsin is not essential for normal protein digestion

because it only acts on approximately 10

–

15% of dietary proteins before it is inactivated in the duodenum, where pH increases due to pancreatic bicarbonate ion

secretion (Lærke and Hedemann, 2012). Therefore, in the stomach, only a limited

mixing of feed occurs and is subject to the action of pepsin, which may partially

degrades the dietary proteins.

1.1.3 Small Intestine

The pig small intestine is a long digestion tube and shaped like a spiral. Its wall

has millions of tiny finger-like protrusions known as villi, which increase the absorptive

surface area. Epithelial cells lining the wall of the small intestine also secrete enzymes

that break down feed particles and facilitate nutrient absorption (Lærke and Hedemann,

2012). The pig small intestine is longer in comparison to those of other farm animals

and permits intensive endogenous digestion under near-neutral conditions (Wenk, 2001).

The first section of the small intestine is known as the duodenum. It is here that liver

and pancreas secretions enter the digestive system, where they are essential for

digestion and absorption of various nutrients utilized in intermediary metabolism. Liver

secretions digest fats, while the pancreatic secretions digest lipids, proteins, and

carbohydrates. The products of digestion, such as fatty acids, amino acids, and glucose,

are absorbed in the jejunum and ileum, which are the second and third sections of the

small intestine. The activity of the enzymes contained in feed may disrupt the natural

process of digestion (Strube et al., 2013).

1.1.4 Large Intestine

The large intestine begins with the cecum. In this part of the digestive system,

undigested feed components such as dietary fibers, lipids with high melting point,

insoluble proteins, and endogenous secretions are fermented by the intestinal microflora

and most of the remaining water from undigested feed is reclaimed back into the

circulation (Wenk, 2001).

While the fermentative capacity of the large intestine in pigs is not comparable to

the rumen-reticulum of ruminants, volatile fatty acids (VFA) such as acetate, propionate,

and butyrate produced during fermentation in the large intestine can supply up to 30%

of the energy requirements of growing/finishing pigs (Rérat, 1985; Yen et al., 1991).

However, fermentative energy production is frequently underestimated because the

formation of methane, hydrogen, and fermentative heat decreases the amount of energy

available to the pig (Grieshop et al., 2001), hence decreasing the efficiency of energy

utilization (Giusi-Perier et al., 1989; Noblet et al., 1994).

The intestinal microflora also depends on the host animal’s diet as the main

source of metabolic substrates. Increasing levels of dietary fiber subsequently increase

the amount of substrate that moves into the large intestine for microbial fermentation,

further promoting the activity of carbohydrate-fermenting bacteria (Haberer et al., 1999;

Diebold et al., 2004). Increasing lactic acid and VFA products cause a decrease in the

digesta pH, which creates unfavorable conditions for the growth of protein-fermenting

bacteria (Smith and MacFarlane, 1997). By limiting microbial proteolysis and

increasing carbohydrate fermentation in the large intestine, some of the most

offensive-smelling compounds emanating from pig production are reduced (Mackie et

al., 1998; O’Connell et al., 2005).

1.2 Pancreatic Secretions in the Pig

1.2.1 Structure of the Pancreas

The pancreas is a major part of the digestive system and has an integral role in

absorption and metabolism (Rinderknecht, 1993). It contains 90

–

95% exocrine tissue and about 2

–

3% endocrine tissue (Brannon, 1990), and exhibits the highest rate of protein synthesis and secretion per gram of tissue than any other organ (Logsdon and Ji,

2013). Acinar (>80%) and ductal cells of the exocrine pancreas form a close functional

unit (Fredirick and Jamieson, 1994). The pancreatic acinar cells synthesize and secrete a

variety of digestive enzymes, water, and electrolytes into the duodenum, while cells

lining the pancreatic duct produce bicarbonate-rich secretions that neutralize the acid

chyme entering the duodenum from the stomach and generate a slightly alkaline

environment in the duodenum for optimum pancreatic enzyme activity (Hegyi et al.,

2011). Acinar cell and duct cell secretions constitute the pancreatic juice.

1.2.2 Enzyme Secretion of the Exocrine Pancreas

The main regulatory pathways controlling exocrine pancreatic secretion are

linked to hormone–hormonal and neural–hormonal interactions of regulatory peptides

such as secretin and cholecystokinin (CCK), as well as neurotransmitters from the gut,

pancreas, and vagus nerve (Cook and Young, 1996; Evilevitch et al., 2003; Chandra and

Liddle, 2014). Enzyme secretion also depends on dietary composition, age, feeding

regimen, and postprandial sampling time (Corring et al., 1989; Makkink and Versiegen

1990). Changes in pancreatic synthesis of individual exocrine proteins in response to

polypeptide hormones or nutritional substrates are summarized in Table 1 (Scheele,

1993). Positive feedback mechanisms appear to exist between nutritional substrates in

the diet and the synthesis of pancreatic enzymes required to digest those substrates.

Basal secretion occurs between meals; ingestion of a meal stimulates secretion for 3–7 h,

depending on the frequency of feeding (Lærke and Hedemann, 2012). Average basal

pancreatic secretion is only 0.5 mL/kg/h in suckling pigs, and there is no increase in

response to milk intake (Pierzynowski et al., 1990). Basal pancreatic secretions are

much higher after weaning and increase by 3-fold after feed intake (Pierzynowski et al.,

1990; Rantzer et al., 1997); the most important of these are proteolytic, lipolytic, and

amylolytic enzymes (Ohlsson et al., 1982).

All proteolytic enzymes are secreted as inactive proenzymes from pancreatic

acinar cells to protect the glands from autodigestion (Lærke and Hedemann, 2012).

Activation of these proenzymes is initiated by a regulatory cascade of enteropeptidases

secreted by the duodenal glands within the walls of the small intestine (Lowe, 1994;

Braud et al., 2012; Figure 2). Proteolysis of trypsinogen to trypsin activates other

Table 1

Regulation of protein synthesis in rat exocrine pancreas

(Scheele, 1993)

↑, increase; ↓, decrease; CCK, cholecystokinin; GIP, gastric inhibitory peptide.

aInsulin effect observed only in diabetic animals.

(Braud et al., 2012)

Figure 2. Cascade of biochemical events starting with proenteropeptidase action.

Enteropeptidase converts inactive trypsinogen into active trypsin, which in turn converts the other pancreatic zymogens—chymotrypsinogen, proelastase, carboxypeptidases A and B, and prolipase—into active enzymes.

digestive enzymes in the alkaline environment (Light and Janska, 1989).

The pancreatic juice contains three lipolytic enzymes, including lipase,

carboxylester hydrolase, and phospholipase A2 (Lærke and Hedemann, 2012). Similar

to proteolytic enzymes, the lipolytic enzymes are excreted in an inactive form and

activated by trypsin. All lipolytic enzymes hydrolyze triacylglycerides to fatty acids,

glycerol, and mono- or di-acylglycerides and, therefore, are the most important enzymes

in the digestion of dietary fats (Jensen et al., 1997).

The luminal phase of carbohydrate digestion by pancreatic α -amylase in the

small intestine applies only to starches (Lærke and Hedemann, 2012), which are the

principal dietary carbohydrate and the single most abundant feed energy in diets for

piglets, growing pigs, and sows, comprising 60

–

70% of the total energy intake (Bach Knudsen et al., 2013). Pancreatic α -amylase, like salivary amylase, cleaves the α -1,4

glycosidic linkages of starch substrates, reducing them to glucose, maltose, maltotriose,

and dextrin (Hizukuri et al., 1996). Alpha-amylase activity in pancreatic tissue is

extremely low at birth and rapidly increases with age. Pigs exhibit adaptive secretion,

sensitive to the type and level of starch and dietary fiber in the diet. Weaned piglets

show a sharp increase in basal and postprandial amylase output after the diet is changed

from milk to a diet high in starch (Aumaitre, 1972; Corring, and Saucier, 1972). The

increase in α-amylase activity is also observed when the daily intake of dietary starch is

up to 400%; total α -amylase activity increases by 2.3-fold in 1

–

2 post-prandial hours (Corring and Chayvialle, 1987; Corring et al., 1989). However, replacing dietary

starches with monomeric carbohydrates such as glucose or dextrose had no influence on

α-amylase activity (Corring, 1977). The replacement of starch by cellulose, straw meal,

or pectin significant reduces total α-amylase activity (Mosenthin and Sauer, 1991;

1993).

1.3 Exogenous Enzymes in Porcine Feeding Programs

The efficiency of nutrient digestibility has been assessed in the absence of

exogenous enzymes (Olukosi and Adeola, 2012; Figure 3). Exogenous enzymes such as

carbohydrases and phytase are used worldwide as additives in non-ruminant diets.

Supplementation of porcine diets with these exogenous enzymes is a proven alternative

strategy for improving animal performance by reducing or eliminating anti-nutritional

compounds presents in feedstuffs, thereby increasing nutrient digestibility and

availability (Suga et al., 1978; Xia, 2000; Omogbenigun et al., 2004; De Lange et al.,

(Olukosi and Adeola, 2012)

Figure 3.

Inefficiency associated with digestion in pigs. The inefficiency (%) is calculated using the percentage digestibility of the nutrients. The inefficiency associated with nutrient digestion without exogenous enzyme can be up to 90% for plant calcium (Ca) and phosphorus (P), whereas nitrogen (N) and energy are used with greater efficiency. AA, amino acid

2010). Enzyme effectiveness in animal nutrition depends on type, source, and level of

supplementation, as well as on the type of diet, animal health, and animal productivity.

Since enzymes are protein catalysis, their activity is also susceptible to variations in pH

and may be hydrolyzed by proteases acting inside the gastrointestinal tract. Successful

enzyme supplementation therefore requires an assessment of the factors affecting

enzyme activity and stability during passage (Strube et al., 2013).

1.3.1 Carbohydrase Supplementation

Cell wall NSPs are the major components of dietary fibers and are composed of

cellulose and non-cellulosic polysaccharides (Figure 4). Table 2 shows NSP and

cellulose levels in various cereals. Pigs obtain energy from fiber but only after microbial

fermentation in the gastrointestinal tract and subsequent absorption of VFA (Urriola et

al., 2012), because they have a limited ability to produce endogenous enzyme to digest

NSP. Although soluble fiber is easily fermented, insoluble fiber is not well utilized by

pigs. The greater the concentration of fiber contained in the diet, the lower overall

energy digestibility becomes.

Carbohydrases such as β-glucanase, xylanase, and cellulase, make a significant

(Paloheimo et al., 2001; Sticklen, 2008)

Figure 4. The plant cell wall. (A) Cell wall composition. A plant cell wall is arranged

in layers and contains cellulose microfibrils, hemicellulose, pectin, lignin, and soluble protein. (B) Schematic representation of the cellulose structure.

Table 2

Mean concentration of non-starch polysaccharides (NSP) in cereals and some legume grains (g/kg of dry matter)

(Broz and Ward, 2007)

contribution to porcine diets by catalyzing the breakdown of indigestible cell wall

components into simple sugars. Addition of dietary carbohydrases disrupts the fiber

matrix embedding digestible carbohydrates, thus increasing accessibility of the

digestive enzymes and improving outcomes in terms of diet conversion indexes

(Bindelle et al., 2011). Many of these biologically active enzymes have been purified

from plants, animals, and microorganisms. A large difference in affinity and turnover is

observed for different enzyme sources with the same substrate; for example, xylanases

share a common arabinoxylan substrate but their affinities and activities differ widely

(Biely et al., 1997; Cuyvers et al., 2011). Exogenous enzyme activity is also influenced

by the pH of the digesta. The optimum pH of most exogenous enzymes is between 4

and 5, but great variation may exist between enzymes from different sources, resulting

in catalytic activity high at both lower and higher pH (Svihus, 2010). For example,

Aspergillus-derived xylanase has an optimum pH between 4 and 6 (De Vries and Visser,

2001), but Streptomyces-derived xylanase functions over a pH range of 3–7 (Ding et al.,

2008). This complicated matrix of conditions will determine the scale and variation of

activity for an enzyme added to the diet and as it passes through the digestive tract. The

efficacy of carbohydrase in pigs may be not the result of improvements in nutrient

digestibility alone (Omogbenigun et al., 2004; Ji et al., 2008), but to changes in

digestive content characteristics, which indirectly affect the physiological status of the

gastrointestinal tract. Hydrolysis of polysaccharides yields oligosaccharides such as

xylose or arabinose that serve as prebiotic substrates which are capable of modulating

microbial activity in the gastrointestinal tract, improving the health of the pig (Pluske et

al., 2002; Kiarie et al., 2007). Such prebiotic substances favor the proliferation of lactic

acid bacteria (Högberg and Lindberg, 2004; Kiarie et al., 2007), therefore inhibiting

pathogen growth by competitive exclusion (Hillman et al., 1995).

1.3.2 Phytase Supplementation

Phytate is the main P-containing constituent of many seeds and tubers, which contain a

P content of 282 g/kg (Maga, 1982). In general, phytates constitute about 1–2% by

weight of many cereals and oilseeds. Approximately 36% (rapeseed meal) to 84%

(wheat bran) of total P in these seeds is present in the phytate-bound form (Broz and

Ward, 2007; Table 3). A possible structure of the phytate molecule (myo-inositol

1,2,3,4,5,6-hexakis dihydrogen phosphate; IP6) when complexed with minerals, protein,

and starch in acidic medium is shown in Figure 5A.

Table 3

Total and phytate phosphorus (P) content in feed

(Broz and Ward, 2007)

The P component of phytate is only partially available to pigs because the

endogenous phytase activity in the small intestinal mucosa is insufficient to

dephosphorylate phytate, leading to poor bioavailability of total P in grains (Hu et al.,

1996). It is possible that the standard dietary calcium (Ca) levels have a substantial

negative influence on phytate degradation by endogenous enzymes (Selle et al., 2009).

To ensure an adequate supply of P, it is necessary to include inorganic P supplements in

the porcine diet, although feces produced by these pigs contain high levels of P.

Continuous application of fermented fertilizer made from pig feces causes a build-up of

soil P, thus increasing the potential for P loss and possible water quality degradation

(Adeola, 1999; Abioye et al., 2010).

The mode of action of phytase is illustrated in Figure 5B. Approximately 2–5

U/mL phytase activity is sufficient to satisfy the dietary P requirement (Golovan et al.,

2001). One way to reduce fecal P content is through supplementation of exogenous

phytase. Inclusion of phytase in porcine diets increases the digestibility of P and

phytate-bound minerals, and enhances protein digestibility (Beers and Jongbloed, 1992),

thus increasing the concentration of IP3–IP5 in the small intestine relative to IP6 (Hu et

al., 1996) and reducing P excretion (Li et al., 1998). Another approach to reducing

(Kies et al., 2001; Yu et al., 2012)

Figure 5. The structure of phytate. (A) A putative phytate structure and its

interaction with protein, starch, and cation. (B) Schematic of microbial phytase activity on dietary phytates. Phytate is numbered according to Agranoff’s nomenclature, with the 2-phosphate axial end pointing upwards, with the carbon atoms numbered anticlockwise around the ring. Arrows indicate the phosphomonoester bonds under attack by the phytase.

phytate is exogenous expression of microbial phytase genes in pigs that seems to

require almost no inorganic P supplementation for normal growth and excrete up to

75% less fecal P than non-transgenic pigs (Golovan et al., 2001); however, the activities

of phytase decrease with age.

Bacterial phytase is typically more effective than fungal phytases in terms of the

amount of P released per unit of phytase (Kornegay and Qian, 1996; Adeola et al., 2004;

Augspurger et al., 2007). The characteristics of phytases from different sources are

listed in Table 4. In general, manufacturer-recommended levels of commercially

available phytases replace inorganic P levels by 0.12% in porcine diets (Jendza et al.,

2006). Increasing amounts of phytase in the diet is associated with a curvilinear increase

in the release of P from phytate (Kornegay, 2001).

Most phytases from microbial sources have optimum working pH values in the

acidic range (Igbasan et al., 2000); the low pH environment in the gastric phase is ideal

for making phytate susceptible to hydrolysis (Greiner and Konietzny, 2006). Therefore,

phytase activity in digesta from the stomach is usually higher than in digesta from the

upper small intestine (Jongbloed et al., 1992) and no phytase activity is detectable in

lower small intestine digesta (Yi and Kornegay, 1996). Low phytase activity is also

Table 4

Common phytase enzymes used in swine nutrition

¹

(Olukosi et al., 2012)

1Other enzymes not listed include proteases and lipases of microbial origin, especially those of Bacillus spp.²Most of these characteristics are applicable only to Escherichia coli phytase.

detected in the intestinal contents of transgenic pigs carrying phytase gene driven by a

mouse parotid secretory protein promoter (Golovan et al., 2001).

1.3.3 Enzyme Combinations

Feed enzymes are available as single-component enzymes or as

multiple-component enzymes for which activity is generated in a single fermentation

(Masey O’Neill et al., 2012). Addition of supplemental enzymes in combination is

expected to produce synergistic effects; however, additional studies of multiple enzyme

combinations are needed to elucidate their potential effect on the typical ingredients

utilized in porcine diets.

Chapter 2 Characterization of a Putative

Pancreatic Amylase Gene Promoter from a Pig

2.1 Objective

Successful genetic engineering strategies require the use of promoters with

appropriate characteristics for the desired objective (Potenza et al., 2004). In the

laboratory, transgenic constructs are often designed with viral promoters and enhancer

sequences to increase the efficiency of expression in most tissues and species (Betrabet

et al., 2004). However, it remains unclear if there are risks associated with human

consumption of integrated viral promoter or enhancer sequences within the transgenic

pig genome. Combining spatial and temporal control of gene expression allows

transgenic livestock production to be more useful in agricultural applications.

Accordingly, the use of tissue-specific and its own promoters for heterologous gene

expression may minimize the negative effects of transgenes on the normal growth and

development of transgenic individuals (Zheng and Baum, 2008). In a previous study

(Lin, 2004), a 2,488-bp 5'-flanking region from a pig whose own pancreatic amylase

gene was cloned by the genomic walking technique; however, its structural features

were not characterized. To characterize this putative promoter, we used the cloning

technique, a green fluorescent protein (GFP) reporter gene, bioinformatics, and

functional analysis in a rat pancreatic tumor cell line.

2.2 Materials and Methods

2.2.1 Computer-based Analysis: the 5'-flanking Region of the Porcine Pancreatic Amylase Gene

A core promoter region was predicted in the 2,488-bp 5'-flanking region of the

porcine pancreatic amylase gene by using the Neural Network Promoter Prediction

program (NNPP) program, version 2.2 (Reese, 2001). Putative transcription factor

binding sites were identified by using the Transcription Element Search System (TESS)

database (http://www.cbil.upenn.edu/cgi-bin/tess/tess), a string-based search tool

similar to local alignment software.

2.2.2 Cloning of the Fluorescent Protein Reporter Construct

The putative promoter region and N-terminal signal peptide-encoding region of

the porcine pancreatic amylase gene (Darnis et al., 1999) were amplified with primers

containing restriction sequences of

NotI

and

EcoRV

(forward-1,

5'-GCGGCCGCCTGACATAAGCTGAA; reverse-1, 5'-GATATCGGCCCAGCAGAA

CCCAA) and with the following cycling conditions: 94°C for 5 min; 35 cycles at 94°C

for 30 sec, 65°C for 30 sec, and 72°C for 2 min; and a final extension at 72°C for 7 min.

Subsequently, the PCR products were digested with designate endonucleases, and

purified with a QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). The digested

and purified PCR product was subcloned into the humanized Renilla reniformis-derived

GFP (phrGFP) mammalian expression vector (Agilent Technologies, Cedar Creek, TX,

USA) to generate the pAMY-hrGFP construct.

2.2.3 Culture of Rat Pancreatic AR-42J Cells

The rat pancreatic tumor cell line AR-42J was purchased from the Food Industry

Research and Development Institute (Hsinchu, Taiwan). Cells were maintained at 37°C

under an atmosphere of 5% CO2/95% air in Ham’s F12K medium (Invitrogen, Grand

Island, NY, USA) containing 1.5 g/L sodium bicarbonate and supplemented with 2 mM

L-glutamine, 20% fetal bovine serum (FBS; Invitrogen), and 1% antibiotic-antimycotic

mixture (Invitrogen). The medium was changed daily and cells were harvested at the

indicated time points.

2.2.4 Transfection and Transient Expression of the AMY-GFP Reporter Construct

For transfection, cells were seeded and cultured in 35-mm dishes to 70–80%

confluence. Attached cells were rinsed with PBS twice, then with serum-free Ham’s

F12K medium supplemented with 50 mM dexamethasone (Raffaniello et al., 2009).

Two micrograms of pAMY-hrGFP were transfected into the cells by using a jetPEI

cationic polymer transfection reagent (Polyplus-transfection, New York, NY, USA).

Non-transfected cells were used as a negative control. At 48 h post-transfection, GFP

expression was observed under an inverted fluorescence microscope (Axiovert 200;

Carl Zeiss, Jena, Germany).

2.3 Results

2.3.1 Characterization of the Putative Porcine Pancreatic Amylase Gene Promoter Region

A DNA fragment containing 2,488-bp of the 5'-flanking region in conjunction

with 45 bp signal peptide-coding sequence from the porcine pancreatic amylase gene

was isolated and sequenced. Bioinformatics analysis indicated that the potential

transcriptional start sites were located 11, 438, and 1,380 bp upstream of the ATG

translational start site, and a TATAAA (TATA-box) sequence was located 43 bp away

(Figure 6). Potential transcriptional factor binding sites in this region were specifically

for insulin promoter factor 1 (IPF-1), glucocorticoid response element (GRE),

hepatocyte nuclear factor 3-beta (HNF3-β), Opaque-2, and prolamin boxbinding factor

(PBF).

2.3.2 In Vitro Activity of the Porcine Pancreatic Amylase Gene Promoter

Amylase promoter activity was examined in AR-42J cells transfected with the

GFP reporter plasmid (Figure 7A). The results showed that this 2,488-bp 5'-flanking

region was sufficient to activate reporter gene expression (Figure 7B).

2.4 Discussion

Digestive gene expression and enzyme activity are modulated in response to

dietary chemical signals (Karasov et al., 2011). Pancreatic enzymes accommodate each

individual’s dietary composition via biosynthesis and secretion of specific enzymes

(Owsley et al., 1986; Simoes Nunes, 1986; Flores et al., 1988); for example, pancreatic

amylase secretion is sensitive to the amount of starch in the diet (Corring and

Chayvialle, 1987; Mosenthin and Sauer, 1993). Low intake of carbohydrate usually

results in a decrease in amylase expression at the transcriptional level (Giorgi et al.,

1984). Fluctuations in amylase activity may be modulated at the level of gene

expression. Therefore, the amylase gene promoter may be useful for gene expression

through dietary stimulate.

Linking molecular mechanisms with physiological functions improves the

understanding of hormonal control of the pancreatic amylase promoter in regulating its

downstream gene expression (Ma et al., 2004; Scheele, 1993). GFP fluorescence was

observed in AR-42J cells transfected with pAMY-GFP, suggesting this construct

contained a functional promoter that was able to drive the downstream gene expression.

Several cis-acting regulatory elements were predicted in the 5'-flanking region of the

porcine pancreatic amylase gene, including IPF-1, GRE, and HNF3-β. These

observations are consistent with the results of previous studies in humans, mice, and sea

bass (Liberzon et al., 2004; Ma et al., 2004). The close physical association of the three

functional elements is consistent with the role of insulin in mediating dietary response

(Hani et al., 1999; Lechner et al., 2001; Lee et al., 2002; Scheele, 1993). However, the

insulin-response element is necessary but not sufficient for the regulation of amylase by

dietary carbohydrate in mice (Schmid and Meisler, 1992). Glucocorticoid signaling also

plays a key role in modulating the expression of many digestive enzymes in the

exocrine pancreas (Kaiser et al., 1996; Logsdon et al., 1985). The hexanucleotide

5'-TGTCCT-3' is important for GRE activity (Slater et al., 1985), and pancreatic

transcription factor (PTF-1) is required for glucocorticoid induction of mouse amylase

expression (Slater et al., 1993). Glucocorticoids such as the synthetic dexamethasone

typically bind the ligand-binding domain of GRE and increase GRE binding activity in

a dose-dependent manner, thus promoting nuclear translocation and up-regulating

amylase gene expression (Logsdon et al., 1985; Pratt etv al., 2004). Two

hexanucleotides were present in the 5'-flanking region of the porcine pancreatic amylase

gene, but no functional PTF-1 binding site had been identified. Instead, a functional

HNF3-β (also known as FoxA2) binding site was found in this region, where it

recognizes an HNF3-binding site required for transcriptional activation of genes in

exocrine acinar cells (Rausa et al., 1997). These observations are consistent with a

previous study in sea bass, which showed that glucocorticoid stimulation of amylase

promoter-driven gene expression is direct via GRE (Ma et al., 2004). Our results also

revealed the presence of binding sites for the corn-transcriptional activators PBF and

Opaque-2 scattered throughout the 5'-flanking region of the porcine pancreatic amylase

gene. These proteins are expressed in parallel in corn endosperm and their putative

orthologs have been identified in a large number of other cereals, including rice, barley,

and wheat (Singh, 1998; Hwang et al., 2004). PBF and Opaque-2 act singly or in

combination to promote transcription of numerous seed storage protein genes during

seed development (Hwang et al., 2004). It remains unknown whether binding of these

putative PBF and Opaque-2 transcription factors regulates the porcine pancreatic

promoter following food intake.

2.5 Summary

We ligated the 2,488-bp 5'-flanking sequence of the porcine pancreatic amylase

gene to GFP and found that this region contained promoter activity and had the

potential to control heterologous gene expression. The structural features of the

promoter include several potential transcriptional factor-binding sites for IPF-1, GRE,

HNF3-β, Opaque-2, and PBF.

Figure 6.

The 5'-flanking region of the porcine pancreatic amylase gene.

Lowercase and uppercase letters represent the 5'-flanking region and coding sequence, respectively. The deduced amino acid sequence is presented below the coding region of exon 1, and the sequence encoding the signal peptide is boxed. Numbers alongside the sequence refer to the nucleotide position relative to the translation initiation site, designated as nucleotide +1. Three putative transcription start sites are indicated in bold with asterisks. The TATA box is highlighted in gray, and putative binding sites for insulin promoter factor 1 (IPF-1, dotted), glucocorticoid response element (GRE, broken), hepatocyte nuclear factor 3-beta (HNF3-β, double), Opaque-2 (thick), and prolamin box binding factor (PBF, thin) are underlined.

Figure 7. Verification of promoter activity. (A) Schematic diagram of the

pAMY-GFP plasmid vector. The construct, consisting of the putative amylase promoter region (black box), porcine pancreatic amylase signal peptide (SP, gray box), and a humanized Renilla reniformis-derived GFP (hrGFP) gene (white box), was designed for transient transfection of AR-42J cells. (B) GFP expression in transfected cells. Cells were maintained in the presence of 50 mM dexamethasone. Negative controls (upper panel) were treated as above, but without transfection reagent or plasmid. After 48 h treatment, bright field (left panels) and fluorescence (right panels) images of the cultured cells were collected at 200× magnification. Scale bars represent 50 µm.

Chapter 3 Evaluation of Transgenic Pigs

Expressing AMY-PHY and AMY-CEL in the Gastrointestinal Tract

3.1 Objective

There are many potential applications of the transgenic methodology in the

development of new and improved strains of livestock (Prather et al., 2008). Practical

applications of transgenics in livestock production include enhanced prolificacy and

reproductive performance (Rejduch et al., 2002), increased feed utilization and growth

rate (Vize et al., 1988), improved carcass composition (Pan et al., 2010), improved milk

composition (Yang et al., 2011), and increased disease resistance (Donovan et al., 2005).

Accordingly, genomic integration of exogenous phytase and fibrolytic genes for direct

expression in the gastrointestinal tract may be a rational and economical choice

approach to reach the reduction of the fecal P and N contents. In a previous study (Lin,

2004), a transgenic founder was generated by co-injection of the pronuclei of fertilized

pig eggs with two constructs in which the bacterial phytase transgene (AMY-PHY) and

fungal cellulase transgene (AMY-CEL) were under the control of the 2,488-bp

5'-flanking region of the pancreatic amylase gene. However, it remained unclear

whether the pancreatic amylase promoter could be used to induce transgene expression

in the porcine gastrointestinal tract. The activity of secreted enzymes in the

gastrointestinal tract has also not been established. The objective of this study was to

evaluate AMY-PHY and AMY-CEL expression by RT-PCR, western blotting, and

specific enzyme activity assays in pancreatic tissue and gastrointestinal tract contents.

3.2 Materials and Methods

All surgical and experimental procedures were approved by the Institutional

Animal Care and Use Committee,National Taiwan University, Taiwan.

3.2.1 PCR and Southern Blot Analyses

Genomic DNAs were isolated from the ear tissue of each newborns pig by using

standard proteinase K-sodium dodecyl sulfate digestion followed by the

phenol-chloroform extraction (Herrmann and Frischauf, 1987). One microgram of

genomic DNA was employed as the template for a PCR reaction. Two sets of primers

were used to amplify the full-length DNA of the transgenes to confirm the presence of

the AMY-PHY (forward-2, 5'-GGATCCCAGAGTGAGCCGGAGCT; reverse-2,

5'-CTCGAGTTACAAACTGCACGCCGGTA) and AMY-CEL (forward-3,

5'-GGATCCATTATGAAACCCGAACCA; reverse-3, 5'-CTCGAGTTATTCCTTTGG

TTTTTC) cassettes in the transgenic pigs versus their nontransgenic littermates.

Southern blotting was performed as previously described (Yang et al., 2004). In brief,

radio-active probes were generated with PCR-amplified fragments and labeled with

[α-³²P]dCTP by using the Amersham Rediprime II DNA labeling system (GE

Healthcare, Fairfield, CT, USA). Two probes were designed to target the structural

genes of the AMY-PHY (forward-2; reverse-4, 5'-TCAGTCACGTTCGCGTTATCT)

and AMY-CEL (forward-3; reverse-5, 5'- TCCGTTCCATTCAACTGGTG) cassettes

and were expected to amplify 431- and 599-bp products, respectively. After

hybridization and variable stringency washing, membranes were subjected to

phosphor-image analysis with a Typhoon 9200 scanner (GE Healthcare).

3.2.2 RNA Extraction and Analyses of RT-PCR

Total RNA was extracted from snap frozen pancreas, heart, liver, lung, kidney,

muscle, stomach, and duodenum tissues of transgenic pigs using an RNeasy Lipid

Tissue Mini kit (Qiagen) with on-column DNase digestion (RNase-Free DNase Set;

Qiagen) according to manufacturer instructions. RT-PCR was performed using a

SuperScript III One-Step RT-PCR System with a Platinum Taq DNA polymerase kit

(Invitrogen). In brief, 50 ng of each purified RNA sample was used as the starting

material. Two primer sets were designed to target the structural genes of the AMY-PHY

(forward-2; reverse-4) and AMY-CEL (forward-3; reverse-5) cassettes, and were

expected to amplify 431- and 599-bp products, respectively. Beta-2 microglobulin

(B2M; forward-4, 5

'

-AACGGAAAGCCAAATTACCTG; reverse-6, 5

'

-GTGATGCCG GTTAGTGGTCTG) served as a positive internal control, yielding a 259-bp fragment.

Reactions were performed as follows: 55°C for 40 min; 94°C for 2 min; 35 cycles at

94°C for 30 sec, 64°C for 30 sec, and 68°C for 2 min; and a final extension at 68°C for

7 min. Amplified products were separated by 0.8% agarose gel electrophoresis and

visualized by EtBr staining.

3.2.3 Protein Extraction

All pigs had body weights in excess of 120 kg prior to sampling. Porcine tissue

samples as well as digesta samples from the duodenum and ileum were collected at least

6 h after feeding and then stored in liquid nitrogen. A piece of frozen porcine pancreatic

tissue was pulverized within liquid nitrogen and its total protein was extracted through

homogenization in ice-cold RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1%

NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor

(Millipore, Billerica, MA, USA) under gently rotation for 2 h at 4°C. The supernatants

from either the RIPA-soluble pancreatic protein or duodenal content preparations were

collected by centrifugation at 13,500 ×g for 20 min at 4°C. Total protein concentrations

were determined with a BCA Protein Assay kit (Pierce, Rockford, IL, USA). All

samples were stored at −20°C until SDS-PAGE analysis.

3.2.4 Cloning and Purification of Recombinant Proteins

Recombinant phytase and cellulase were cloned using a pET-52 3C/LIC cloning

kit (Merck, Darmstadt, Germany) according to the manufacturer instructions. Primer

sets were used to generate the full-length phytase coding region (forward-5,

5'-CAGGGACCCGGTCAGAGTGAGCCGGAGCT; reverse-7, 5'-GGCACCAGAGC

GTTTTACAAACTGCACGCCGGTA) and cellulase coding region (forward-6,

5'-GACGACGACAAGATATTATGAAACCCGAACCA; reverse-8, 5'-CTCGAGTTA

TTCCTTTGGTTTTTC) with N-terminal Strep-tag II and C-terminal 10×-His

tag-specific sequences (underline). The resulting fragments were ligated into the

pET-52b(+) 3C/LIC vector (Merck) and transformed into BL21(DE3) competent cells

(Stratagene, La Jolla, CA, USA), respectively. Transformants were selected for

kanamycin resistance and resulting clones were sequenced to confirm the insert

orientation. Each selected clone containing the recombinant plasmid pET52-CEL was

grown in 4 mL LB medium before being transferred to 200 mL Overnight Express

Instant TB Medium (Merck) supplemented with 1% glycerol and 1 µg/mL kanamycin.

The expressed His-Strep-tagged protein from bacterial lysates was purified by using a

Ni-NTA Spin kit (Qiagen) followed by Strep-Tactin Magnetic Beads (Qiagen)

according to manufacturer instructions. Fractions from each step of purification were

fractionated on 10% SDS-PAGE following Coomassie blue staining. Purified

recombinant protein was quantified using a BCA protein assay reagent (Bio-Rad,

Hercules, CA, USA).

3.2.5 Western Blotting Analyses

Protein samples (50 µg) were boiled for 10 min in 5× sample buffer (60 mM

Tris-HCl [pH 6.8], 25% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.1%

bromophenol blue) and loaded onto an 8% polyacrylamide gels (TGX Acrylamide

Starter Kit, Bio-Rad). After electrophoresis, the proteins were electrotransferred to

Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare) using the

Trans-Blot Turbo Transfer System (Bio-Rad). Blocking was performed in Tris-buffered

saline with Tween 20 (TBS-T; 20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1%

Tween-20) containing 10% skim milk overnight at 4°C before incubating 2 h in our

custom-made rabbit polyclonal antiserum generated against recombinant cellulase

(1:2,000; GeneTex, Hsinchu, Taiwan) or phytase (1:2,500; GeneTex) at room

temperature with gentle agitation. Membranes were then washed with TBS-T and

incubated with horseradish peroxidase (HRP)-conjugated chicken anti-rabbit IgG

(1:20,000; Abcam, Cambridge, UK) for 1 h at room temperature. All primary and

secondary antibodies were diluted in 5% skim milk in TBS-T. Bound horseradish

peroxidase (HRP)-conjugate was detected using a Western Lightning Plus ECL kit