Biotechnology and Genetic
Engineering
Ana Carolina Amaral Coutinho
Introduction
 Growth Hormone
Factor controlling growth in vertebrates.
Factor controlling of metabolism in mammals.
 Human Growth Hormone
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Single polypeptide chain;
191 amino acids;
Molecular Mass 22 kDa;
Presence of:
2 disulfide bonds
4 α-helices
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 Growth Hormone Deficiencies (GHD)
Due to pituitary disease, adenoma, trauma, and primary deficiencies.
The symptoms of GHD is mainly cardiovascular disease leading to:
Premature death;
Lower bone density;
Increased fat mass;
Increased protein lipids.
Actual effective treatment to GHD:
Human Growth Hormone Replacement Therapy
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 Human Growth Hormone Replacement Therapy
1958 – First use of hGH therapy
Use of human cadaver pituitaries
1980’s – Use of human cadaver pituitaries were terminated
They were linked to CJD (Creutzfeldt-Jakob)
1985 – Recombinant Human Growth Hormone
Development of new strategies
Objectives
• Expression of a recombinant human growth hormone in the mammary glands of
transgenic rabbits at the transcription and the translation levels;
• Verification of the stability of the transgene transmission;
• Mapping of the transgene by fluorescence in situ hybridization (FISH).
Materials and Methods
1.
2.
3.
4.
5.
Sample collection;
Screening of transgene and sequencing;
Mapping of trangene;
mRNA expression level;
Immunoradiometric assay.
Materials and Methods
1. Sample collection;
2.
3.
4.
5.
Screening of transgene and sequencing;
Mapping of trangene;
mRNA expression level;
Immunoradiometric assay.
1. Sample Collection
 Recombinant WAP:6xHishGH vector
Containing:
- Rat WAP promoter;
- Histidine tag;
- Thrombin recognition site;
- Entire genomic sequence of hGH.
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• Transgenic rabbit (N0. 61) generation
Founder Male Rabbit F0
Microinjection of a gene construct
• After reaching the sexual maturity:
Collect of sperm followed by artificial insemination of non-transgenic females.
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• Transgenic rabbit (N0. 61) generation
Founder Male Rabbit F0
Microinjection of a gene construct
• After reaching the sexual maturity:
Collect of sperm followed by artificial insemination of non-transgenic females.
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• Transgenic rabbit (N0. 61) generation
Founder Male Rabbit F0
Microinjection of a gene construct
• After reaching the sexual maturity:
Collect of sperm followed by artificial insemination of non-transgenic females.
Females resulted from F1
were also inseminated
with sperm from
transgenic male
Materials and Methods
1. Sample collection;
2. Screening of transgene and sequencing;
3. Mapping of trangene;
4. mRNA expression level;
5. Immunoradiometric assay.
2. Screening of transgene and sequencing
• Total DNA extracted from tissue ear bioptats.
• PCR Screening:
First PCR Screening: 313-bp DNA fragment amplification.
5′-Cy5-AGTCTTCCTCCTGTGGGTC- 3′
5′-TCTCTCTCCATCCCTCCAG-3′
Second PCR Screening: 524-bp DNA fragment amplification
5′-Cy5-GTCCCAACC- CAACCATTC-3′
5′-TGGCGATACTCA- CATTCAGA-3′
• PCR products were fractionated in 6% polyacrylamide gel.
Materials and Methods
1. Sample collection;
2. Screening of transgene and sequencing;
3. Mapping of transgene;
4. mRNA expression level;
5. Immunoradiometric assay.
3. Mapping of transgene
3. Mapping of transgene
• Mapping
Performed by FISH (Fluorescence In Situ Hybridization) using primary cell cultures
of skin fibroblasts.
• Analysis of the karyotype
Performed using G-banding of metaphase chromosomes.
DNA probe specific for the transgene was labeled with biotin-dUTP and purified on
a Sephadex G-50 column.
For signal detection, cell spreads were incubated with antibodies labeled with
fluoresceine.
Materials and Methods
1. Sample collection;
2. Screening of transgene and sequencing;
3. Mapping of transgene;
4. mRNA expression level;
5. Immunoradiometric assay.
4. mRNA expression level
• RNA isolation and analysis
Exfoliated mammary epithelial cells collected from milk from lactating transgenic
females.
Tissue biopsies such as brain, heart, kidney, liver, and salivary gland which have
ectopic expression of the transgene.
Cells of non-transgenic animals as a control.
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Detection of rhGH mRNA
By reverse transcriptase reaction;
Materials and Methods
1.
2.
3.
4.
Sample collection;
Screening of transgene and sequencing;
Mapping of transgene;
mRNA expression level;
5. Immunoradiometric assay.
5. Immunoradiometric assay
• Detection of [ ]
Performed in milk samples collected from transgenic and non-transgenic lactating females.
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Analysis
Samples of whole milk, defatted fraction, casein fraction, and whey fraction were
separated
rhGH concentration.
Results
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The founder male No. 61 transmitted the transgene to the progeny.
Production of transgenic females and males.
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F1 Males Heterozygotes
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F1 Females Heterozygotes
F2 Males/Females Homozygotes
Presence of transgene in an expected size was confirmed by PCR analysis of
DNA from ear samples from transgenic rabbits.
By sequencing analysis
Transgene in F1 animals was 100% identical to a sequence of the gene construct
used for the generation of founder animal No. 61.
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Screening data provided from PCR to
analyze the transmission of rhGH
Results

FISH Mapping
Discrimination heterozygous and homozygous animals.
All rabbits with integrated transgene = transgene in the q26-27 telomere region of
chromosome 7
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
Recombinant human growth hormone detected:
- Total unfractionated milk samples;
- Casein;
- Whey fraction from transgenic females.
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rhGH was not found in the serum samples of all transgenic animals.
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Milk fractions from wild type animals were negative for the presence of rhGH.
The concentration of rhGH in milk samples observed for the transgenic homozygote rabbits was between 10 and 12 μg/ml.
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
Presence of rhGH mRNA was demonstrated in exfoliated mammary epithelial
cells.
Consistent evidence for the presence of
rhGH RNA in mammary gland cells was
obtained.
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The transgene was not ectopically expressed in the brain, heart, kidney, liver, and
salivary gland.
Discussion
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The mammary gland as the best available bioreactor.
Source of a variety of complex recombinant proteins.
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Constructed gene showed no interference with normal rabbit development.
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The transgene WAP:6xHishGH became stably integrated into the host genome;
Being transmitted to the offspring and expressed.
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rhGH mRNA is specifically translated in the mammary gland and the product
secreted into the milk as biologically inactive protein.
Mammary epithelial cells expressed the transgene.
Reference
1. Daniel Lipinski & Joanna Zeyland & Marlena Szalata & Andrzej
Plawski & Malgorzata Jarmuz & Jacek Jura & Aleksandra Korcz &
Zdzislaw Smorag & Marek Pienkowski & Ryszard Slomski. 2012.
Expression of human growth hormone in the milk of transgenic rabbits
with transgene mapped to the telomere region of chromosome 7q. J
Appl Genetics 53 (2012) 435–442.