Transgenik biyoreaktörlerde rekombinant proteinlerin üretimi

Transgenik hayvanlar kendi genomunda başka bir organizmaya ait rekombinant bir geni taşıyan hayvanlardır. Mikroenjeksiyon tekniği ile üretilen ve daha çok hastalık modellemesi amacıyla geliştirilen transgenik farelerin ilki 1980 yılında yayınlanmıştır. Fakat, özellikle transfer edilen genin ürününün elde edilmesi, yani, transgenik hayvanların bir çeşit "protein üreten fabrika" olarak kullanılması hedeflendiğinde bu alandaki çalışmalar da farklı bir boyut kazanmıştır. Temel amaç, elde edilecek ürünün ki bu ürün terapötik değere sahiptir ve tıbbi açıdan son derece değerlidir- bol miktarda ve mümkün olduğunca saf olarak kazanılmasıdır. Aktarılan rekombinant genin (transgenin) ekspresyonunun, promotor gibi gen ekspresyonunu kontrol edici dizilerin yardımıyla transgenik hayvanın belirli dokularına yönlendirilmesi ve böylece hedef proteinin süt ve idrar gibi vücut sıvılarından salgılanmasının sağlanması çalışmaları, çok az süt verdiklerinden dolayı farelerin bu amaç için uygun olmadığını açıkça ortaya koymuştur. Varılan nokta, rekombinant proteinlerin üretilmesinde kullanılacak transgenik hayvanların, bol süt veren çiftlik hayvanlarından seçilmesi gerekliliği olmuştur. Tekniğin ilk adımında, mezbaha materyalinden veya ovaryumlardan oosit pick-up (OPU) ile elde edilen olgunlaşmamış oositlerin in vitro maturasyonu ve in vitro fertilizasyonu gerçekleştirilmektedir. Daha sonra, pronükleer dönemde bulunan zigotlara mikroenjeksiyon yöntemi ile 2-4 ng/ul konsantrasyonda gen konstraktından (transgen) 2 piko litre verilmektedir. Süt alınması temel şart olduğundan, in vitro kültür ortamında morula ya da blastosist aşamasına gelen embriyolara sex tayini yapılarak dişi olanlar belirlenir. Sonraki adım, di-şi embriyoların taşıyıcı annelere transfer edilmeleridir. Doğan yavrular arasından transgenik olanlar çeşitli moleküler biyolojik yöntemlerle (PCR, Southern Blot) saptanır. Transgenik çiftlik hayvanlarının üretimi ile yılda 1 tona yakın rekombinant protein, ölü insan dokularındakinden çok daha saf, hijyenik ve bol miktarda elde edilebilmektedir.

Production of recombinant proteins in transgenic bioreactors

Transgenic animals carry a foreign recombinant gene in their genomes. The first transgenic mice which were produced by pronuclear DNA microinjection for modelling some human diseases have been reported in 1980. However, when the target is especially to use these transgenic animals as protein producing factories and to obtain a recombinant product of transgene in large amounts, all transgenic studies would gain a special point of view this time. The main purpose in this area is to produce recombinant proteins having medical and commercial value in large amounts. In these studies, transgene is expressed in certain tissues such as mammary cells with assistance DNA control elements called promotor. Thus, the targeted recombinant protein can be obtained from the transgenic animal's milk or urine. Under these circumstances, it would be clear that a transgenic mouse's milk amount is not enough for this aim. Because of that, transgenic animals which will be used for producing recombinant proteins in their milk must be choosen from among the livestocks. In the first step of this technique, in vitro maturation and in vitro fertilization of unmatured oocytes obtained from the ovaries which were recovered from the slaughterhouse were performed. After that, 2 pico litres of gene construct (transgene) in 2-4 ng/ul concentration is transferred into the pronucleus of zygotes. By the sex determination of morula or blastocyst stage embryos, the females are identified to guaranty to get milk yield. The next step is to transfer the female embryos into the foster mosters. Transgenic pups are determined by some molecular techniques such as PCR and Southern Blot. About one tone of recombinant protein is obtained in a year and these proteins much more pure and hygenic than obtained in dead human tissues.

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