will gm farm animals play a role in future food production · pdf filemammary gland against...

• Challenges to global food production

• Brief survey of methodological background and technologies

• Current status of GM animals for food production

• The plea for targeted and diversified animal production

Will Will GM GM farmfarm animalsanimals playplay a a rolerole in in futurefuture

foodfood productionproduction ??

Institute of Farm Animal Genetics (FLI)

Mariensee, Neustadt, Germany

Prof. Dr. Heiner Niemann

Mistra Biotech Symposium „Breeding genetically modified animals for food production, Uppsala, Sweden| June 24th, 2014

Evolution of farm animal breeding

• Domestication

• Proliferation of „useful“ populations

• Selection according to the Exterieur

• Selection according to specific traits

• Systematic breeding based on population genetics and statistics

• Reproductive technologies (AI, ET, IVP, etc.)

• Molecular genetics and genome based breeding concepts(specific sequences, SNPs, etc.)

Domestication of pigs

Sus scrofaGerman Landrace

• Loss of pigmentation, shorter skulls

• ~30% less brain volume, less sensitive sensory organs (nose, ears),

• less weight of inner organs (heart, kidneys, liever, etc.)

• Different body composition.

Basic structure of todays globalizedfarm animal breeding

Proven

Top-

genetics

Multiplication units

Production farms

(Meat, Milk, Eggs)

Calculation of the breeding value

• Progress in breeding value still is mainly achieved via the paternal side.

• The breeding value is calculated via the performance of the offspring (f. ex. 3-4000 inseminations per test bull).

• Globally, breeding of the main populations is currentlybeing switched to the genomic breeding value.

• The breeding value contains several parameters.

• Breeding values are globally available.

Milk production/cow and no. of dairy cows in USA (1940-1995) and Germany (1800-2005)

Roberts, 2001

1800 850 ltr/year

1913 2200 ltr/year

1950 2500 ltr/year

1967 3687 ltr/year

1983 4733 ltr/year

1996 5513 ltr/year

2000 6000 ltr/year

2005 6800 ltr/year

2012 7500 ltr/year

GermanyUSA

Qualitative improvement of animal production: Fat contents in pork

Projected increase in human global population

Selected constraints of agriculturalproduction

• ~5% of global land is usable for agriculture

• Increase in affluency if several parts of the world is associated

with changes in diet towards more valuable animal proteins

• Environmental constraints of livestock production (~1/3 of climate

relevant emission comes from agriculture)

• FAO: 1.3 kg CO2eq/milk (Northern America, Europe);

7.5 CO2eq/milk (Africa)

• Consequences: Food production needs to be doubled or tripled

• Need: higher productivity without detrimental side effects

(sustainable intensification).

��

Projected increase in global demand for meatand meat products 1993 - 2020

51%

6%

7%

6%

16%

14%

China & Südostasien

südl. Asien

westl. Asien & Nordafrika

südl. Afrika

Lateinamerika

westl. Länder

China & South East Asia

Southern AsiaWestern Asia & Northern Africa

Southern Africa

Latin America

Western countries

Roberts, 2001

Nature 426, 2003

A new era in biology: Genome sequencing, somatic cloning and embryonic stem cells

2004: first draft of bovine and chicken genome

2006: dog, bee

2009: cattle, horse

2012: pig

2014: sheep

1997

1998

Sequencing and annotation of thebovine genome

• The bovine genome has ~22,000 genes, similar to humans and other mammals (http://BovineGenome.org);

• Size of the genome: 2.87 (Gbp) Giga base pairs, 60 chromosomes;

• ~5% of the genome are transcriptionally active;

• High degree of homology (14,345 orthologous genes) of the bovine genome with human, dog, rat, mouse;

• Transposons and ruminant-specific repeats;

• Bovine-specific variation in genes related to lactation, reproduction, energy efficiency and the immune system.

Reduction of cost for genome sequencing

Nature 497, 2013

The practical use of this new genomicinformation

• Better targeted breeding programmesGenomic breeding values; Direct sequencing

• Transcriptomics/Proteomics/Phenomics

• Production of transgenic animals

• New knowledge on genetic diversity

• Descent studies

• Comparative genomics

Production of transgenic animals bysomatic cell nuclear transfer

Transgenic cell

Transgenicanimal

Cloning vector with

transgene

Somatic cells

(fibroblasts)Cloning,

restriction

In vitro culture in

selection medium

Cells with inte-grated

transgene

Oocyte donor

analysis,

copy number

Transfection

Metaphase II

oocyte

Removal of

chromosomes

Aspiration

Enucleated

oocyte

Recipient animal

Embryo transfer

In vitro culture

Delivery of cloned calfTransfer of cell

into ooplasm

Advantages and disadvantages of microinjection and somaticnuclear transfer (SCNT) for the production of transgenic animals

Microinjection SCNT

Integration efficiency: + +++

Integration site: random random/targeted

Gene-knock-out: - +++

Construct size: >50 kb (Art.Chr.) ~30-50 kb

Technical requirem.: ++ +++

Chimerism: +++ -

Expr. screen. in vitro: (+) +++

Expression pattern: variable controlled, consistent

Multi-Transgenics: + +++

Timeline for Transgenics: long immediate availability

New tools for the productionof transgenic animals

• DNA-Nucleases

(Zinc finger nucleases (ZFNs), Transcription activator-

like effector nucleases (TALEN), Clustered regulatory interspaced short palindromic repeats (CRISPR/Cas9)

• Transposons (SB, PiggyBac, Tol 2)

• Pluripotent , reprogrammed cells

electro-

poration

ZFN plasmids

Cell culture of

porcine fetal

fibroblasts (WT)

Gal-epitope

DNA-isolation

- Cel-I assay

- NHEJ

FACS analysis

with cells

(IB4-FITC)

- biallelic KO

Selection for

Gal-negative

cells with

magnetic

beads

SCNT

Gal-negative piglets

Cell culture:

Gal-positive

and Gal-

negative cells

Strategy for the production of pigs with a homozygous knockout for -Gal

Left: First pig with an ZFN-induced homozygous KO of an endogenous gene

(Liliy, born 20.12.11, had to be euthanized) Right: Lia (born 06.01.11, still alive)

Three ZFN GalKO piglets born on

14.04.11

c

Piglets cloned from cells with a homozygousgene-knockout mediated by specific ZFN

Hauschild et al. PNAS 108, 12013 – 12017, 2011

Application perspectives for transgenicfarm animals

•Agricultural perspectivesGrowth and developmentWool productionLactation (amount, composition)Disease resistance (Mx-gene, IgA, BSE)Environmental ImprovementsDietetic ImprovementsReproduction

•Biomedical perspectivesGene Pharming Humane blood substituteXenotransplantationInhibitors of chemical weapons

•Basic researchDisease modelsReprogramming models

Transgenic animals with improved milk production

Introduced modification Application Species Reference

Bovine ɑ-lactalbumin Increase milk yield Pig Wheeler et al.

and piglet survival 2001

Bovine b-and k-casein Improved milk Cattle Brophy et al.

composition 2003

siRNA-ß-lactoglobulin reduced allergenicity Cattle Jabed et al.

2012

β-lac-GFP-neo- improved udder Cattle Wall et al. 2005

L ycostaphin health

hlysozyme in ß-cas locus improved udder Cattle Liu et al. 2014

health

Brophy et al. 2003, Nature Biotechnology 21, 157-162

Production of transgenic cattle with elevatedconcentration of ß- and k-Casein in milk

Production of ß-lactoglobulin free milks in siRNA-ß-lac transgenic cows

Jabed et al. 2012, PNAS 109, 16811-6

miRNA-mediated depletion of BLG in bovine milk

Jabed A et al. PNAS 2012;109:16811-16816

Targeted microRNA expression in dairy cattle directsproduction of β-lactoglobulin-free, high-casein milk

Cow Milk Total Casein, mg/g Whey, mg/gɑ-Lac BLG-A BLG-b Total

miRNA 6-4 Induced, day 1 98.2 3.9 0.0 0.0 3.9




WT-1 Natural, day 69 39.6 1.5 5.7 0.6 7.8

WT-2 Induced, day 5 38.8 1.5 7.6 0.7 9.8

WT-3 Induced, day 5 32.5 1.5 7.3 0.9 9.4

WT-4 Colostrum, day 1 48.1 1.7 10.1 4.0 15.7

SEM-* - 1.27 0.09 0.12 0.11 0.12

Jabed A et al. PNAS 2012;109:16811-16816

Wall et al. 2005, Nature Biotechnol., 23, 445-451

Resistance against St. aureus infusions: Tg: 18/21(85.3%)

(Infection only with very high Doses) vs. Non-tg 13/47 (31.7%)

Transgenic cows with Lysostaphin mediated resistance of themammary gland against infections with Staphylococcus aureus

Mastitis resistant cows by transgenic expression of human lysozyme expression from the ß-casein locus

Liu et al.,Proc. R. Soc. B. 281, 2014

Transgenic animals for improved meatproduction


Insulin-like growth factor 1 Increased meat Pig Pursel et al. production 1999

Human and porcine growth Increased meat Pig Pursel et al.

hormone releasing factor production 1990

Draghia-Akli

et al. 1999

Human growth hormone Increased meat Sheep Rexroad et al.

releasing factor production 1989

Bovine, human and porcine Increased meat Pig Pursel et al.

growth hormone production 1989, 1990

Nottle et al. 1999

Ovine growth hormone Increased meat Sheep Ward and Brown

production 1989

Adams et al. 2002maP2-FAD 2 less fat in pork Pig Saeki et al. 2004

pCAGGS-hfat-1 less fat in pork Pig Lai et al. 2006

Constructs mMTI-bGH* hMTI-pGH(cDNA)**

Weight gain + 23 10 - 20%

Feed efficiency + 18 10 - 15%

Backfat thickness 7.5 mm significantly

(from 21 mm) reduced

„Side effects “ + + + - (30 - 40ng/ml GH)

Improvements in economically importantparameters of growth hormone transgenic swine

*Pursel et al. 1990; **Seamark and Nottle (BresaGen)

mMTImMTI--hIGFhIGF--II transgenictransgenic pigspigs

Large loin mass: + 30%

Carcass lean tissue: + 10%

Total carcass fat: - 20%

Improvements in economically importantparameters of transgenic swine

Pursel et al (1999)

Saeki et al.(2004), PNAS 101, 6361-6366

• Unsaturated fatty acids play a major

role in membrane structure of cells and

as precursors of prostaglandins,

leukotriens and glycolipids.

• Mammals lack the enzyme desaturase.

Thus they cannot produce linolic acid

(18:2n-6) and α-linolenic acid (18:3n-3)

• Plants (f.ex. spinach) are a rich source

for these poly-unsaturated fatty acids.

• Spinach desaturase transgenic pigs

have a 10-fold higher content of poly-

unsaturated fatty acids in fat tissue.

• Pork with an increased level of poly-

unsaturated fatty acids may contribute

to healthier food in human consumption.

Spinach desaturase expressing pigs

n-3 and n-6 fatty acids concentration and n-6/n-3 ratios in tail samples from hfat-1 transgenic and wild-type piglets

Fatty acids in tails Transgenic piglets (n = 8) Wild-type piglets (n = 8)

ALA (18:3 n-3, %) 0.94 ± 0.10 0.63 ± 0.04

EPA (20:5 n-3, %) 4.21 ± 0.60 0.26 ± 0.07

DPA (22:5 n-3, %) 1.69 ± 0.19 0.35 ± 0.05

DHA (22:6 n-3, %) 1.75 ± 0.23 0.95 ± 0.21

Total n-3 FA (%) 8.59 ± 0.84 2.18 ± 0.25

Total n-6 FA (%) 14.28 ± 1.31 18.46 ± 1.41

n-6/n-3 ratio 1.69 ± 0.30 8.52 ± 0.62

Lai et al. 2006, Nature Biotech

Growth hormone transgenic salmon is as safe as conventionally producted salmon (FDA 2012)

GH transgenic salmon and its wildtype counterpart (Aquabounty)

Transgenic animals with improved fibreproduction


Ovine insulin-like growth 6.2% more fleece Sheep Damak et al. factor 1 1996

Ovine growth hormone Improved wool Sheep Adams et al.

production 2002

Ovine keratine intermediate Improved wool Sheep Bawden et al.

filament processing and 1998

wearing properties

Bacterial serine transacety- Improved wool Sheep Ward 2000

lase and O-acetylserine production

sulfhydrylase

Approaches towards animals transgenic forenhanced disease resistance

•• General:General:

• Mx 1 protein (pigs)

• Immunoglobulin A (pigs)

• Visna virus envelope (sheep)

• Transmissible Gastroenteritis virus (TGEV; mammary glandspecific mouse model)

• Prion knock-out (cattle)

• siRNA mediated knockdown of pathogenic virus expression

•• MammaryMammary glandgland::

• ɑ-Lactalbumin (pigs)

• Lysozyme (goats, cattle): antimicrobial effects

• Lactoferrin (cattle): bacteriostatic, bacteriocidic, iron provider

• Lycostaphin-transgenesis: St. aureus resistant cows

Pigs fed hLZ- milk have improved fecal consistency and activity scores

Cooper CA, Garas Klobas LC, Maga EA, Murray JD (2013)

PLoS ONE 8(3): e58409. doi:10.1371/journal.pone.0058409http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058409

Milk from hLZ transgenic goats helpschildren with diarrhea

Cloned cattle with resistance against BSE by knock-out of the prion-locus

Richt et al., Nature Biotechnology 25, 132- 138, 2007

Inhibition of PERV expression in porcine fibroblasts

via RNA-interference

Ø Selection of effective siRNA, inhibition in PERV-infected human cells(Karlas et al., 2004)

Ø Using lentiviral vectors and primary pig fibroblasts (Dieckhoff et al., 2006)

LTR EF-1alfa

cPPT

GFP WPRE tetO H1 SIN

siRNA

loxP

LTR EF-1alfa

cPPT


siRNA

loxP

siRNA

loxP

cPPT

LTR EF-1alfa

cPPT


siRNA

loxP

LTR EF-1alfa

cPPT


siRNA

loxP

siRNA

loxP

cPPT

LTR EF-1alfa

cPPT


siRNA

loxP

LTR EF-1alfa

cPPT


siRNA

loxP

siRNAcPPT

LTR EF-1alfa

cPPT


siRNA

loxP

LTR EF-1alfa

cPPT


siRNA

loxP

siRNA

loxP

cPPT

LTR EF-1alfa GFP WPRE tetO H1 SINLTR EF-1alfa GFP WPRE tetO H1 SINLTR EF-1alfa GFP WPRE tetO H1 SINLTR GFP WPRE tetO H1SIN

shRNAcPPT

EF1-α

5‘ LTR

loxP

3‘ LTR

LTR H1 SIN hPGK GFP WPRE SIN

siRNAcPPT

LTR H1 SIN hPGK GFP WPRE SIN

siRNA

cPPT

LTR H1 hPGK GFP WPRE SIN

shRNA

cPPT

5‘ LTR 3‘ LTR

pLVTHM

RRL-PGK-G

FP

pLVTHM

ß-actinp27Gag c

ontro

l

RRL-PGK-G

FP-pol2

pLVTHM-pol2

contro

l

sorte

d29 d

116 d

171 d

relative PERV expression

[%]

0

20

40

60

80

100

120

140 SE101

contro

l

RRL-PGK-G

FP-pol2

RRL-PGK-G

FP

contro

l

contro

l

plVTHM-pol2

SE105 P1 F10

RRL-PGK-GFP

Generation of transgenic pigs expressing PERV-specific siRNA

6 transgenic animals carrying the vector expressing pol2-siRNA were born Sept. 9, 2006

Ø Vector integration in all animals (a)

Ø Expression of specific siRNA in all tissues (b)

Ø Inhibition of PERV expression in several tissues over 2-3 years (c)

1 2 3 4 5 6 + -

Transgenic piglets

0

20

40

60

80

100

120

C 1 2 C 1 2 C 1 2 C 1 2 C 1 2

heart lung liver kidney muscle

% e

xp

ressio

n

lun

g

sple

en

liv

er

kid

ne

y

pa

ncr

ea

s

he

art

PK

15

po

l2

PK

15

TH

M

PK

15

RN

Ase

con

tro

l

con

tro

l

(a)

(b)

(c)

CD46-KO by ZFN targeting to create resistance against ESF

• Exon 5 coding for CCP3 (complement control protein)

• Mutations in exon 5 HUS

Hawkins and Oliaro. FEBS Lett. 2010;584:4838-44.

Fremeaux-Bacchi et al. 2006. J Am Soc Nephrol. 17:2017-25

• Resistance against swine fever?

• Cells will be analyzed at Isle of Riems

CD46-KO by ZFN targeting to create resistance against ESF

Electro-

poration

ZFN plasmids

PK15 or

SK6 cell

culture

CD46

FACS (CD46-

antibody-FITC )

Selection of CD46-

negative cells with

magnetic beads

Cell culture:

CD46 positive

and CD46

negative cells

Analysis

�Biallelic-

KO rate

Various transgenic animals in foodproduction

Feed conversion

Bacterial Bacterial isocitrate lyase Increased glucose supply Sheep Ward 2000and malate synthase

Human glucose transporter 1 and Improved glucose utilization Fish Krasnov et al. 1999

rat hexokinase II

Phytase expression Improved P-metabolsim Pig Golovan et al. 2001

Adaptation to new habitat

Piscine antifreeze protein Fish farming in colder waters Fish Hew et al. 1999Wang et al. 1995

Golovan et al., Nat. Biotechol. 2001, 19, 741-745

Transgenic swine expressing Phytasein the salivary gland

Expression of Expression of phytasephytase in in thethe

salivarysalivary glandgland of of transgenictransgenic

pigspigs

Reduction of anorganicphosphorus feeding via improved metabolism

Reduction of phosphorusexcretion by up to 75%

Reduction of costs

Environment protection

Golovan et al. (2001), Nature Biotechnol. 19, 741-745

Expression of Phytase in the salivary gland of transgenic pigs

Transgenic farm animals: Status inthe FDA registration process

A brief history of some of the genetically engineered food animals submitted to

the US Food and Drug Administration (FDA) for review. No such animals has yet

been approved.

Animal Purpose Created History

Salmon Grows to market size faster than

conventional salmon

1989

(Massachusetts)

1995 FDA receives application

2008 Fish farm moved to Panama

2010 Cleared by FDA scientific advisory panel

Pig Produces more milk to nurse

healthier young

1993

(Illinois)


Goat Milk has human lysozymes to treat

diarrhoel disease

1999

(California)

2003 Funding denied by USDA

2008 FDA receives appplication

2011 Research moved to Brazil

Pig Efficiently digests plant

phosphorus,

R educing pollution

1999

(Ontario,

Canada)


2012 Pigs killed owing to lack of commercial

interest

Cow

Sheep,

goat, pig

Increased muscle mass without

reduced fertility

2010

(Texas)


Nature 490,318-319, 2012

Genetic Engineering News 22, September 2002

Cloned cattle with an additional chromosome

PerspectivePerspective: : DiversifiedDiversified and and targetedtargeted livestocklivestock

productionproduction

• „Agricultural" livestock breeding and production

• Diversified milk production (total demand, specifications)

• Diversified meat production in sufficient amounts

• Egg production

• Niche production

• Rural conservation

• Aquacultures

• "Biomedical" livestock production• Gene Pharming

• Xenotransplantation (cells, tissues, organs)

• Disease models

• Reproduction models (f.ex. therap. cloning, ARTs)

• „Ecological" livestock production (extensification)

• Sports animals/companion animals/pets

The future: Targeted and diversified dairy production

• Full fat normal milk

• Defatted milk (knock-out of key lipid enzymes)

• Curd production (enhanced casein expression)

• Cheese production (enhanced casein expression)

• Coffee whitener and Creme liquor (-casein)

• Hypo-allergenic milk (reduced or omitted -lactoglobulin)

• Lactose-free or –reduced milk (-lactalbumin knock-out, additional lactase expression)

• Infant milk (enhanced lactoferrin expression)

• Improved udder health (lysozyme, etc.)

• Pharmaceutical proteins

Evolution of farm animal breeding

• Domestication

• Proliferation of „useful“ populations

• Selection according to the Exterieur

• Selection according to specific traits

• Systematic breeding based on populationgenetics and statistics

• Reproductive technologies (AI, ET, IVP, etc)

• Molecular genetics (sequence information, SNPs, etc)

• Somatic clones, stem cells, transgenics

Summary and conclusions

• The genomes of most farm animals are sequenced and annotated. Methods for genomic analyses (transcriptomics, proteomics, phenomics, etc.) will increasingly become available for all agricultural species.

• Transgenic farm animals can now be produced by a variety of technologies. SCNT in combination with Designer nucleases (ZFNs, TALEN, CRISPR/Cas) is good option to efficiently produce animalswith targeted genetic modifications.

• There are already numerous examples for genetically modifiedanimals with useful application perspectives in food production.

• These novel technologies are valuable tools for the development of a diversified and targeted dairy cattle production.

• Widespread application and public acceptance of these newdevelopments critically depend on the agricultural frame work (EU, WTO) and a careful consideration of the potential consequences foragricultural production structures.

Just the Beginning

Thank you for your attention.

will gm farm animals play a role in future food production · pdf filemammary gland against...

Documents