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Journal of Biotechnology 124 (2006) 469–472 High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow Daniel Salamone , Lino Bara˜nao , Claudio Santos , Leonardo Bussmann , Jorge Artuso , Carlos Werning , Aida Prync , Cesar Carbonetto , Susana Dabsys , Carlos Munar , Roberto Salaberry , Guillermo Berra , Ignacio Berra , Nahuel Fern´andez , Mariana Papouchado , Marcelo Foti , Norberto Judewicz , Ignacio Mujica , Luciana Mu˜noz , Silvina Fen´andez Alvarez , Eliseo Gonz´alez , Juan Zimmermann , Marcelo Criscuolo , Carlos Melo a Biosidus S.A., Buenos Aires, Argentina b Facultad de Agronom´ıa, Universidad de Buenos Aires, Buenos Aires, Argentina c Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina d Instituto de Biolog´ıa y Medicina Experimental-CONICET, Argentina e Instituto Nacional de Tecnolog´ıa Agropecuaria, Argentina f Munar y Asociados, La Plata, Pcia de Buenos Aires, Buenos Aires, Argentina g Centro de Diagn´ostico Molecular S.A., Buenos Aires, Argentina Received 15 September 2005; accepted 4 January 2006 Abstract
Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biophar- maceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Herewe report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels ofup to 5 g l−1. The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological andsomatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable ofproducing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for theproduction of this hormone.
2006 Published by Elsevier B.V.
Keywords: Recombinant hGH; Transgenic cloned cow; Animal biotechnology ∗ Corresponding author at: Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina. Tel.: +54 11 4786 2564; fax: +54 11 4786 2564.
E-mail address: (L. Bara˜nao).
0168-1656/$ – see front matter 2006 Published by Elsevier B.V.
D. Salamone et al. / Journal of Biotechnology 124 (2006) 469–472 The expression of proteins with potential thera- Mature oocytes were denuded by vortexing for 3 min peutic applications in the milk of livestock species in PBS with 1 mg ml−1 bovine testis hyaluronidase.
appears to be one of the most attractive commer- Metaphases were assessed and oocytes were enucleated cial applications of animal transgenesis by visualization with Hoechst 33342 (5 ␮g ml−1) under UV light (<6 s). Donor cells at G0/G1 stages were fused to proteins required in large amounts that cannot be to enucleated oocytes by an electrical pulse. After 3 h, obtained cost-effectively in current bioreactors, such activation was induced by incubation in TL-HEPES with 5 ␮M ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and 48 g l−1 have been reported for the production of and cultured in SOF medium with an atmosphere of these proteins in the milk of sheep and cows, respec- 5% CO2 + 5% O2 + 90% N2. Development to blasto- cysts (days 7–9) was recorded. One or two blastocysts The present study was set up to analyze the feasibil- per recipient cow were transferred non-surgically, and ity of using the milk of transgenic cattle as an alternative pregnancies at 30 days or 60 days were determined by for the commercial production of human growth hor- Cleavage rates and blastocyst production were not Recombinant hGH was produced in E. coli culture significantly different between the different cell lines following the procedure routinely used for the com- and control fibroblasts. A total of 15 transgenic calves mercial production of the hormone. A 650 bp cDNA were born (4 from cell line Leo 0 and 11 form line encoding the hGH was cloned into a pBST plasmid Leo 2, while line Leo 1 produced no pregnancies). The under the control of a phage RNA polymerase pro- presence of the transgene in DNA isolated from the moter. The expression of the gene in the resultant transgenic calves blood was verified by PCR amplifi- construct is inducible with IPTG. One milliliters of cation of DNA sections comprising part of the promoter bacterial suspension from the original culture was used transgene and the entire coding region. Only one calf to generate a subsequent culture in a New Brunswick from line Leo 0 showed the complete sequence for Scientific IF 250 bioreactor. Bacteria were grown dur- the hGH coding region and the ␤-casein promoter, ing 12 h and then induced to produce hGH for 3 h.
whereas partial deletions in the 3 end of the transgene They were subsequently collected by cross-flow fil- were observed in the other animals. This is proba- tration (Filtron equipment) and disrupted by high- bly a consequence of the heterogeneity of the trans- pressure treatment (Rannie equipment). Material was genic cell lines, since they were not subjected to clonal centrifuged and the supernatant passed through an immunoaffinity column (Affigel-10 Bio-Rad Laborato- Lactation was induced in transgenic animals by ries resin + anti-hGH monoclonal antibodies). Recom- a biphasic treatment. The first phase of the treat- binant hGH was eluted and fully purified by RP-HPLC ment involved the combined sc administration of (C4) and further molecular exclusion chromatogra- estrogens (estradiol benzoate, Histeren®, Instituto Rosenbusch) and progestagens (medroxyprogesterone For the production of cloned transgenic cows, fetal acetate, Pronal®, Aton), consisting of five successive fibroblasts were obtained from a 75-day-old Jersey applications of each drug, in a dose of 0.1 mg kg−1 BW fetus and transfected with two separate plasmids, one and 0.25 mg kg−1 BW, respectively, every 48 h (i.e., containing the human growth hormone gene under the on days 1, 3, 5, 7 and 9, assuming that the treatment control of a bovine ␤-casein promoter, and the other commences on day 1). The second phase included the bearing a neomycin resistance gene. After selection for sc administration of dexamethasone (Decadron, Sidus) 14 days with 800 ␮g ml−1geneticin, several colonies and oxytocin (Orasthin®, Hoechst Marion Roussel). A were isolated. Three cell lines were obtained from those total amount of 20 mg of the former was injected over a colonies: Leo 0, Leo 1 and Leo 2, which were used as period comprising days 18–20 (one-third of total mass being administrated each day), and three applications Oocytes were aspirated from slaughterhouse ovaries of 50 IU of the latter were given on days 21–23. Milking and matured in TCM-199 + 5% FCS at 39 ◦C for 24 h.
D. Salamone et al. / Journal of Biotechnology 124 (2006) 469–472 Fig. 1. Bioactive hGH levels in the milk of the transgenic clonedcow.
Bioactivity of hGH in the milk, measured by a Nb2 lymphoma cell bioassay (startedto increase from values around 2 g l−1 at the onset oflactation and rose steadily reaching values of 5.0 g l−1(A highly significant correlation was observedbetween hGH bioactivity and its immunoactivity, asdetermined with a specific antibody. Milk hGH levelsshowed three peaks (30, 60 and 80 days after induc-tion), which were associated with the onset of estrouscycles.
Fig. 2. Coomassie blue staining (panel A) and Western blot (panelB) of bacterial extracts and milk from the transgenic cow producing Human GH was also detected in the cow serum, hGH. Lane 1, molecular weight marker; Lane 2, bacterial extract reaching a maximum of 3000 ng ml−1 and then lev- before immunoaffinity chromatography; Lane 3, bacterial extract eling at around 600 ng ml−1. Since the presence of after immunoaffinity chromatography; Lane 4, bacterial extract after hGH in serum was observed 3 weeks before the onset final purification; Lane 5, whey milk before immunoaffinity chro- of lactation, our interpretation is that rather than to matography; Lane 6, whey milk after immunoaffinity chromatogra-phy; Lane 7, whey milk after final purification.
an ectopic expression of the transgene, the circulat-ing hGH may derive from leakage from the mam-mary gland due to a non-vectorial secretion before comparison, in the cell extract from E. coli hGH rep- lactogenesis II. In fact, tight junctions between adja- resented less than 5%. shows a Western blot of cent mammary secretory cells do not develop until the same samples, which was carried out with a mono- just prior to parturition, and hence, proteins that are clonal antibody against hGH. A faint band correspond- constitutively synthesized by the developing secretory ing to hGH cleavage products could be observed, being epithelial cells are secreted into the interstitial fluid, the proportion of cleaved products similar between bac- and ultimately find their way into serum. This mecha- terial cell extracts and bovine milk serum. Peptidic nism was described for the milk proteins ␣-lactalbumin mapping of hGH from milk gave an identical result to that of the hormone produced by bacterial culture Analysis of the whey milk by SDS-PAGE and In order to initiate a production herd, we have cloned Coomassie blue staining showed a major band corre- two calves from ear fibroblasts of the founder cow. PCR and Southern blots analysis showed a genomic profile represents about 10% of the total protein content. As identical to that of the donor cow. In addition, after D. Salamone et al. / Journal of Biotechnology 124 (2006) 469–472 standard procedures of superovulation and artificial References
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