Mammary Gland as Bioreactor

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Mammary Gland as Bioreactor

• By
• Chase Turner and Sarah Bogolin

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Why the mammary gland?

• Less expensive than protein production of cells
  in culture
• Similar method to the method of transgenic mice
• Generally has no ill effects on either lactating
  females or nursing progeny
• Many different proteins can be expressed
• Proteins in milk do not occur in blood making the
  mammary gland an ideal bioreactor

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General method of mammary gland as bioreactor

• Vector with Your Favorite Gene under control of
  mammary gland specific promoter
• Injected into pronucleus of goat ovum
• Implanted into foster mother
• Transgenic progeny identified by PCR
• Milk collected from transgenic animal
  fractionated to isolate the pure YFG product

PBIO450AnimalGeneticEngLecture08
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Human lactoferrin in the milk of goats

• Gaining a high level of expression of protein
• Use of replication-defective adenoviral vectors

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Human lactoferrin

• Lactoferrin is iron-binding glycoprotein,
• it exhibits many useful biology functional
  activities
• such as antibacterial, antifungal, antiviral,
  antitumor,
• anti-inflammatory and immunoregulatory
  activities
• Also considered an important component of the
  non-specific immune system
• May represent a novel therapeutic with broad
  spectrum potential

Cartoon diagram of recombinant human lactoferrin.
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Method used to gain high expression of protein

• Direct infusion into the mammary gland via the
  teat canal
• Used to diminish high cost and to gain certain
  results of protein
• Faster than transgenic system
• Useful tool for large scale production of
  recombinant proteins of biopharmaceutical interest

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Materials and Methods

• Human embryonic kidney (HEK) cells cultured
• Primary goat mammary epithelial (GME) cells
  isolated from early lactation goats
• Mammary parenchymal tissue finely minced and
  digested overnight
• Centrifuged, filtered only epithelial cells
  remain

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Materials and Methods

• PCR synthesis of human lactoferrin (hLTF) cDNA
• PCR product digested with HindIII and EcoR V
  enzymes
• Cloned into vector pcDNA3.1 (named p3LTF)
• pIR-G (plasmid containing GFP)
• Two plasmids cloned to become plasmid pLTF-GFP
• Subsequent transformations yielded the
  replication-defective adenovirus vector pAD-hLTF

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• Adult female goats
• GME cells cultured
• Cells infected with recombinant adenovirus
  Ad-hLTF
• 72 hours after infection, cells harvested and
  assayed for rhLTF
• Adenovirus infused
• Viral vectors directly infused into the left
  mammary gland of goats

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• rhLTF purification from milk
• Treated goats milked on identical days
• Milk collected and centrifuged
• Supernatant milk separated from layers
• This was precipitated with 55 ammonium sulfates
• Precipitated proteins were collected and verified
  by SDS-PAGE and Western blot

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SDS-PAGE and Western blot

• Results confirmed no specific protein was found
  in normal GME cells
• Lane 1 is the infected cells confirming
  infection of GME cells
• Lane 2 non infected cells
• Lane 3 rhLTF

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Adenovirus vector and virus preparation

• Contains an internal ribosome entry site sequence
• Allows the expression of two different genes of
  interest at high levels
• GFP gene
• hLTF gene
• Both genes under control of CMV promoter

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GFP protein as live marker

• To facilitate the optimal virus harvest time
• Efficacy of transduction in vivo or in vitro
  studies
• Intense GFP fluorescence indicated a high
  expression level of rhLTF in this study

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Effectiveness of pAd-hLTF vector in vivo

• Human lactoferrin expression in treated glands
  increased gradually
• Peaked at day 6
• Then dropped dramatically
• SDS-PAGE analysis showed no consistent difference
  in the protein profiles from the infused glands
  and the control glands

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Concentrations of viral vector tested

• Amount of rhLTF in milk directly dependent of
  injected adenoviral vectors
• Expression peak reached at day 6 of lactation
• However, treatment of 2x109 PFU/ml adenoviral
  vector did not improve expression level of rhLTF
• Similar expression patterns observed in other
  studies

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• Lane 1 milk serum of infected pAd-hLTF goats
• Lane 2 milk serum protein precipitated with 55
  (NH4)2SO4
• Lane 3 serum protein at pH 4.6 by HCL
• Lane 4 and 5 standard weight and rhLTF protein
• Lane 1 standard rhLTF
• Lane 2 serum from uninfected glands
• Lanes 3-6 serum of infected glands from day 2 to
  5 post-infusion

The rhLTF purification from the milk of goats.
For Coomassie blue-stained SDSPAGE (10) gel
Expression of rhLTF in the milk of goats. Western
blot of milk serum from a goat mammary gland
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Why similar expression pattern?

• Possibly because
• Death of infected epithelium cells due to the
  cytopathic effects associated with adenovirus
  infection
• Strong cytotoxic response of the host immune
  system against infected cells

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What does this Study Show?

• Demonstrated that a high expression level of hLTF
  can be achieved in goats( above 2 g/L)
• Methods established are simple, fast, and could
  be more effective for production of human
  lactoferrin and other biopharmaceutical and
  nutritional proteins
• Recombinant adenovirus does not incorporate
  itself into the genome of the host cell
• This results in only transient expression of the
  human lactoferrin gene in mammary gland
  epithelial cells.

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Summary

• Direct transduction of goat mammary epithelium
  cells with adenovirus vector could be a less
  expensive and simpler alternative for the
  production of recombinant proteins in milk.
• Availability of large amounts of recombinant
  human lactoferrin should greatly further
  subsequent research and increase its potential
  applications in medicine.