Transgenic Live-Bearing Fish and Crustaceans Produced by Transforming Immature Gonads with Replication-Defective Pantropic Retroviral Vectors

Abstract

Transgenic animals have been routinely produced by microinjecting or electroporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However, these techniques are inapplicable to live-bearing fish and many crustacean species for which unfertilized or newly fertilized eggs are not readily obtainable. In the present study, replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R) or beta-gal) were used to directly transform the immature ovary or testis of a live-bearing fish (Poeciliopsis lucida) and crayfish (Procambarus clarkii). The fraction of the progeny derived from these treated individuals shown to contain the neo(R) reporter gene by an assay based on polymerase chain reaction (PCR) was significant. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. Expression of reporter genes was detected by a reverse transcription-nested PCR assay. These results showed that transgenic live-bearing fish and crustaceans could be easily produced by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.

Life stages and reproductive components of the Marmorkrebs (marbled crayfish), the first parthenogenetic decapod crustacean

“…Moreover, parthenogenesis and easy accessibility of the eggs for genetic manipulations are ideal premises to create transgenic laboratory strains that would open new windows in many fields of research on crustaceans. An appropriate method for creation of transgenic crayfish has been developed by Sarmasik et al (2001), who successfully transferred a reporter gene into the oocytes of Procambarus clarkii using a retroviral vector technique. Hence, the marbled crayfish seems to be a good candidate for becoming the first transgenic laboratory decapod or even the "Drosophila" of the Decapoda.…”

IDENTIFIKASI IKAN CUPANG (Betta imbelis) TRANSGENIK FOUNDER MEMBAWA GEN PENYANDI HORMON PERTUMBUHAN; Identification of Transgenic Founder Betta Fish (Betta imbelis) Carry Growth Hormone Gene.

“…Pada penelitian terdahulu untuk B. imbellis yang diintroduksi gen GFP didapatkan individu positif F-1 sebesar 20%-33,33% (Kusrini et al, 2012). Keberhasilan metode transfeksi wild betta menunjukkan nilai yang lebih rendah jika dibandingkan penelitian Sarmasik et al (2001a;2001b) terhadap ikan Poeciliopsis lucida. Berdasarkan penelitian tersebut, Sarmasik et al (2001a;2001b) melaporkan bahwa keberhasilan pewarisan transgen pada keturunan hasil persilangan individu F-0 transgenik x non-transgenik adalah sebesar 45%-70%.…”

“…Hal tersebut kemungkinan bahan reagen yang digunakan berbeda dan kondisi telur juga berbeda. Pada penelitian Sarmasik et al (2001a;2001b) menggunakan metode retroviral vector dan ukuran telur ikan yang lebih kecil sehingga korion lebih tipis.…”

Aplication of Gene Transfer in Aquaculture

“…, 1997, dan sukses menghasilkan transgen. Baru-baru ini juga Sarmasik et al (2001) telah berhasil memproduksi ikan transgenik dengan menyuntukan vektor tersebut ke daerah sekitar gonad ikan gapi (Poecilia lucidai) dan crayfish (Procambarus clarkii). Lu et al (2002) juga berhasil membuat ikan silver sea bream transgenik dengan menyuntikkan cDNA (hormone pertumbuhan ikan rainbow trout dengan promoter ikan mas -actin) yang dicampurkan dengan liposom ke gonad ikan, dan cara ini disebut sebagai "testis-mediated gene transfer".…”