Chronic heat stress induces the expression of HSP genes in the retina of chickens (Gallus gallus).

Introduction: Chronic heat stress during summer is a major challenge imposed by global warming. Chickens are more sensitive to heat stress than mammals because they lack sweat glands. Thus, chickens are more susceptible to heat stress during summer than other seasons. Induction of heat shock protein (HSP) genes is one of the primary defense mechanisms against heat stress. Tissue-specific responses exhibited by different classes of HSPs upon exposure to heat stress have been reported previously in different tissues including the heart, kidney, intestine, blood, and muscle, but not in the retina. Therefore, this study aimed to investigate the expression levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in the retina under chronic heat stress. Methods: This study was conducted during the summers of 2020 and 2021 in Kuwait. Chickens (Gallus gallus) were divided into control and heat-treated groups and sacrificed at different developmental stages. Retinas were extracted and analyzed by using Real Time quantitative Polymerase Chain Reaction (RT-qPCR). Results: Our results from the summer of 2021 were similar to that from the summer of 2020, regardless of whether GAPDH or RPL5 was used as a gene normalizer. All five HSP genes were upregulated in the retina of 21-day-old heat-treated chickens and stayed upregulated until 35 days of age, with the exception of HSP40, which was downregulated. The addition of two more developmental stages in the summer of 2021 showed that at 14 days, all HSP genes were upregulated in the retina of heat-treated chickens. In contrast, at 28 days, HSP27 and HSP40 were downregulated, whereas HSP60, HSP70, and HSP90 were upregulated. Furthermore, our results showed that under chronic heat stress, the highest upregulation of HSP genes was seen at the earliest developmental stages. Discussion: To the best of our knowledge, this is the first study to report the expression levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in the retina under chronic heat stress. Some of our results match the previously reported expression levels of some HSPs in other tissues under heat stress. These results suggest that HSP gene expression can be used as a biomarker for chronic heat stress in the retina.

Retinal Gene Expression of Selective Genes and Histological Stages of Embryonic and Post-Hatch Chickens (Gallus gallus).

Chickens are excellent models for the study of retinal development and function. Gene expression at the correct time is crucial to retinal development and function. The present study aimed to investigate retinal gene expression and morphology in locally grown chickens at various developmental stages. RNA was extracted from the retina at the embryonic and post-hatch stages, and the retinal layers were stained with haematoxylin and eosin (H&E). RT-PCR and RT-qPCR were used for gene expression analysis of 14 selected genes. The results showed that all the retinal genes were expressed at different developmental stages. However, there were slight noticeable variations in expression patterns. At the morphological level, all retinal layers were well observed, except for the outer plexiform layer that became visible in the fifteen-day chick embryo. The current study provides a baseline for standard retinal gene expression of 14 genes and retinal histological staining. The selected genes have different roles in retinal development and function, and most of these genes are associated with retinal diseases. The results obtained here can be applied to molecular retinal research and retinal diseases with genetic factors in retina animal models or human diseases.

Comparing and Optimizing RNA Extraction from the Pancreas of Diabetic and Healthy Rats for Gene Expression Analyses.

Advanced differential gene expression analysis requires high-quality RNA. However, isolating intact pancreatic RNA is challenging due to abundant pancreatic ribonucleases, which limits efficient downstream gene expression analysis. RNAlater treatment reduces endogenous ribonucleases effects through either pre-organ excision via organ mass or bile duct direct injection or organ mass injection post-isolation. We compared RNA extraction protocols to establish a reproducible and effective pancreatic RNA extraction method to obtain high RNA integrity number (RIN) values from healthy and streptozotocin (STZ)-induced diabetic rats for gene expression analyses. Different methods were tested focusing on RNase activity inhibition using RNAlater (Qiagen) pre-harvest of the pancreatic tissue, and extracted RNA quality and concentration were analyzed using NanoDrop spectrophotometer, Agilent Bioanalyzer, and RT-PCR. Inclusion of several pre- and post-excision modifications in the RNeasy Mini Kit (Qiagen) protocol resulted in RIN values more than two-fold higher compared to those using the standard protocol. Additionally, RT-PCR amplification of the housekeeping gene, ß-actin, revealed no differences in extracted RNA quality from healthy and STZ-induced diabetic rats. We compared and developed a more effective and reproducible pancreatic RNA extraction method from healthy and diabetic rats, which resulted in RNA of superior quality and integrity and is suitable for complex molecular investigations.

Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.