The experimental work of this study was carried out in poultry farm of the Department of Animal Production, Faculty of Agriculture, Benha University, Egypt with cooperation of Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City and Life Science Institute University of Michigan, USA from 2013 to 2014.
The present work was carried out to study:
I. Applicability of the modern biological technology for molecular genetics and genetic engineering to isolate, cloning and sequencing of chicken IGF-1 gene:-
IGF-1 gene isolation and cloning from chicken (cobb 500 broilers) as a high producing exotic strain in growth traits and sequencing analysis compared to sequence reference which published in ncbi to transfer it to some local chicken strains.
II. The effect of strain and IGF-1 gene transfer using SMGT techniques on some semen characteristics:-
A total of 400 hens and 96 cock from Mandarah and Silver Montazah strains (200 hen and 48 cock in each strain) were used in IGF-1 gene transfer by zero, 5, 10 and 15 micro gram dose. Semen traits (Individual motility, live sperm and abnormal sperm percentage) were studied in different groups. Then, this semen was used in artificial insemination and egg collected with different groups to incubation.
III. The effect of strain and IGF-1 gene transfer using SMGT techniques on incubation and after incubation traits:-
Fertility, hatchability, early and late embryonic mortality and abnormal chicks percentage were studied in different incubation groups. A total of 2000 chicks were produced from incubation program and growth traits, carcass traits, hormonal profile, histological study in ileum and thyroid gland and rate of calcium absorption studied in pure and transgenic chicks.
Results obtained could be summarized as follow:
I. Applicability of the modern biological technology for molecular genetics and genetic engineering to isolation, cloning and sequencing of chicken IGF-1 gene:-
1. Total RNA was extracted from the chicken cobb-500 liver samples which was the active part in the total DNA in somatic cell.
2. RNA was reverse transcribed into first strand cDNA to complete other steps for IGF-1 gene isolation.
3. Specific primers which complementarities with sequence IGF-1 gene and plasmid structure were used to amplify fragment from total cDNA.
4. Amplification of cIGF-1 from cDNA using specific primers by polymerase chain reaction (PCR).
5. cIGF-1 product was cloned with pCR3.1 vector between Hind III and EcoR1 in restriction site and insertion IGF-1 gene into plasmid mapping using legation enzyme.
6. Plasmid containing IGF-1 gene transformed into E.Coli bacterial colony as a template to increase amount of modification plasmid.
7. The transformed cells were grown and plasmid extraction from E.Coli bacterial colony using Miniprep plasmid.
8. The recombinant plasmid (pCR3.1-cIGF-1) was sequenced using T7 common sequencing by the Sanger sequencing method at DNA sequencing core (life science institute, University of Michigan, USA, http://seqcore.brcf.med.umich.edu) and amino acid sequences translated from the cDNA sequence were compared with sequences in the Gen Bank public database, by using Bioedit software v7.1 which obtained that plasmid containing chicken IGF-1 gene.
II. The effect of strain and IGF-1 gene transfer using SMGT techniques on some semen characteristics:-
1. Before semen incubation, the effect of strain was highly significant (P |
