Transgenic Animals and Lactation

Application of transgenic technology to lactation

Introduce new proteins to give specific changes– Animal growth & health– Bacteria levels– Processing properties– Fat content/type– Amount of protein

UCD first to demonstrate the functional and physical properties of milk can be altered by

the transgenic approach

Expressed human lysozyme in mouse milk

– Made lysozyme at 0.4 mg/ml under the control of the bovine αs1-casein gene

Followed lactation curve

– Faster rennet clotting time and stronger curd

– Slowed growth of mastitis-causing and cold spoilage bacteria Maga et al. 1994 Transgenic Res 3:36-42

Maga et al. 1995 J Dairy Sci 78:2645-2652Maga et al. 1998 J Food Prot 61:52-56

What about a real animal?Non-bioreactor transgenic animals for lactation

Source Promoter Gene SpeciesU. Illinois

M. Wheeler 2002 bα-lac bα-lac Pig2005 bα-lac hIGF-I Pig

UC DavisE. Maga 2004 gβ-lg rSCD Goat

2006 bαs1 hLysozyme GoatUSDA

B. Wall 2005 oβ-lg Lysostaphin Cattle

New ZealandG. Laible 2003 bβ-CN bβ/κ-Casein Cattle

Hypothesis:Animal Growth & Health

Faster growth– Greater pre-weaning gain

Maintenance of growth during illness– Improved disease resistance, immune

factors

Transgenic pigs and lactation

More α-lactalbumin– To increase milk production

Transgenics make more lactose (and milk) and pigs grow faster

IGF-1– Intestinal development

Increased musocal weight and enzyme activity

Noble et al. 2002 J Anim Sci 80:1090-1096Monaco et al. 2005 Transgenic Res 14:761-773

Hypothesis:Fat Content/Type

Short chain, saturated fatty acidsIf enzyme added to modify existing fat:– Change ratio of “bad” fat to “good”

Milk more healthy for human consumptionNovel flavorsNovel products

Stearoyl-CoA desaturase in the mammary gland

The type of fat in milk is modified

– Transgenic goats make less SFA and more MUFA and PUFA

Total amount of fat remains the same

– Transgenic goats make more CLA

Disease resistance implications

Reh et al. 2004 J Dairy Sci 87:3510-3514

-10

-5

0

5

10

15

20

25

30

#34 #37 #53

SFA

MFAPUFA

Percentage change in milk fat composition

Hypothesis:Processing Properties/Amount of

Protein in Milk

If protein with positive charge is made in milk:– Increase protein interactions

Promote faster RCTIncrease gel strengthAffect micelle size

– Novel processing properties– Novel products

If more protein was made in mammary gland:– More food for animal and human consumption– Increase cheese yield/lactation– Modify processing properties– Benefits producers and processors

Transgenic β- and κ-casein cows

To influence milk processing and overall protein levels

– 11.2 mg/ml κ-casein– 15.8 mg/ml β-casein– Altered κ-casein:total

casein ratio– Color change– Amount of fat and

minerals also affected

Brophy et al. 2003 Nat. Biotechnol. 21:157-162

Hypothesis:Animal Growth & Health

Improved disease resistance– Mastitis– Scours

Transgenic dairy cattle and mastitis

Lysostaphintransgenic cows

– Bacterial-derived hydrolase

– Mastitis challenge:71% control glands infected14% transgenic glands infected

– Effective against S. aureus only!

Wall et al. 2005 Nat. Biotechnol. 23:445-451

Hypothesis:Bacteria Levels

Types of bacteria in milk– Cold-spoilage– Lactic acid– Mastitis-causing

If an antimicrobial is made in milk:– Decrease overall level of bacteria in milk

-Stop/slow cold spoilage of milk-Stop/slow outbreaks of human illness from milk

– Affect cheese making???– Slow/stop infection in udder

Mastitis protection

Human lysozyme transgenic goats

Potential benefits:

– Healthier udder

– Changes in processing properties

– Anti-microbial activity

Expressed in goat milk at levels of 270 µg/ml

– 68% of human milk

20 kDa14 kDa

HLZ

Maga et al. 2006 J Dairy Sci 89:518-524

Anti-microbial activity:Improved animal/udder health

Somatic Cell Count (cells/100ml)

458,000*284,000All

609,000409,0003rd

474,000208,0002nd

351,000291,001st

HerdTransgenicParity

P<0.05

Processing properties

Decreased rennet clotting time

Increased curd strength

Implications for cheese manufacturing, types of cheese that can be made

Curd Strength

0102030405060

0 5 10Applied Stress (Pa)

Cur

dSt

reng

th

Anti-microbial activity:Lysozyme goat milk slows the growth of

some bacteria

Transgenic milk was bacteriostatic

– E. coli– S. aureus– P. fragi– S. cohnii– Corynebacteria spp.

Transgenic milk has increased shelf life

Maga et al. 2006 Foodborne Pathog Dis 3:384-392

Transgenic vs Additive

Inhibition of E. coli growth

-0.2

0

0.2

0.4

0.6

0.8

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Reduction in Bacterial Growth

Added LysozymeAdded LactoferrinTransgenic

Antimicrobial activity:Potential to improve human health

Upper Lower Transgenic-Fed 28-8 190 10 28-9 140 20 28-16 7,400 1,100 28-17 80 1,400 Control-Fed 28-10 3,900 5,600,000 28-12 6,600 17,700 28-13 61,000 3,980,000 28-14 590,000 320

Pig Intestinal coliforms (CFU/gram)

Maga et al. 2006 Transgenic Res 15:515-519

Future Work

Desaturase expression

Generation of lactoferrin transgenic goats– First lactation started last week!

Impact of lysozyme milk on:– Mastitis– Processing – Disease prevention– Gut development

Artemis the Goat