To create a reporter knock-in mouse, a target vector construct containing a LacZ gene, neomycin gene, and HSV-tk is introduced into ES cells via electroporation. The ES cells are then screened and genotyped to confirm homologous recombination. Generated ES cells are injected into blastocysts that are implanted into a surrogate mother to generate chimeric mice. Analysis of reporter gene expression is carried out via X-gal staining of tissue from Foxk2+/LacZ, Foxk2LacZ/LacZ, and wild-type mice. Fluorescent immunohistochemistry can further validate results.
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
Understanding of Models use for biomedical research who have similar physiological function like humans ,and the how to generate and which models are useful
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
Understanding of Models use for biomedical research who have similar physiological function like humans ,and the how to generate and which models are useful
Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maizePGS
This lecture was a part of Plant Genetics Seminars - PGS 2017/2018 at Assiut University. These seminars organized by Dr. Ahmed Sallam, Department of Genetics, Faculty of Agriculture, Assiut University
Abstract
Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.
Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maizePGS
This lecture was a part of Plant Genetics Seminars - PGS 2017/2018 at Assiut University. These seminars organized by Dr. Ahmed Sallam, Department of Genetics, Faculty of Agriculture, Assiut University
Abstract
Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.
Agile methodologies for innovative software development projectsSilvia Fragola
How to select the right projects to apply agile methodologies? One size does not fit all! I'd like to share with you some advices based on my experiences. Enjoy the reading!
Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile el...Kalyani Rajalingham
Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperm,
By
Kalyani Rajalingham
Somayeh Haji Kazem Nili
A comparison predictive and adaptive approach - Main differences - When to apply an agile approach? - When a predictive one? - A comparison between PMP® and PMI-ACP® - How to choose between them?
Female mammals achieve dosage compensation by inactivating one of their two X chromosomes
during development, a process entirely dependent on Xist, an X-linked long noncoding
RNA (lncRNA). At the onset of X chromosome inactivation (XCI), Xist is up-regulated
and spreads along the future inactive X chromosome. Contextually, it recruits repressive
histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is
tightly coupled to differentiation and its expression is under the control of both pluripotency
and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate
at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist
regulation in differentiating ES cells, linked to its control and prevention of spurious
transcription factor interactions occurring within Xist regulatory regions. Our findings have a
broader relevance, in the context of complex, developmentally-regulated gene expression.
XCI is a dosage-compensation mechanism that evolved to equalize expression levels of x-linked genes in female (2x) and male (1x) by transcriptional silencing of one x-chromosome in female mammalian cells.
XIC
It is responsible for initiating X inactivation in cis: an X-chromosome fragment that carries a Xic can become
inactivated, whereas one in which the Xic is missing cannot.
The Xic is also involved in ‘counting’, whereby only a single X is kept active per two sets of autosomes in a cell, and all other Xic-carrying chromosomes are inactivated.
Genomic conflict-It arises when genes inside a genome are not transmitted by the same rules
Genes that cause such genomic conflict are called selfish genetic elements (also selfish DNA, ultra-selfish genes, genetic parasites) and can be harmful to the individual.
So selfish gene can be defined as stretches of DNA (genes, fragments of genes, noncoding DNA, portions of chromosomes, whole chromosomes, or sets of chromosomes) that act narrowly to advance their own interests—in other words, replication at the expense of the larger organism.
Here it also presented about what is genomic conflict, types of it, cytoplasmic inheritance, its relation with genomic conflict, ABC model, Molecular mechanism of CMS, Pollen hypothesis, ATP hypothesis, etc.
Conversion of fibroblasts to retinal cells by transcription (final)Lucas Man
A presentation introducing my research project on the conversion of fibroblasts to retinal cells. This presentation summarizes my plans for the project.
1. REPORTER KNOCK-IN MOUSE
BY KALYANI RAJALINGHAM
In order to create a reporter knock-in mouse, one must first create a knock-in construct, and
introduce them into ES cells via methods like electroporation. The target vector construct
would include a 5’-arm, a 3’-arm, a LacZ gene, a neomycin gene, and HSV-tk (Figure 5).
Homologous recombination between the target vector, and the wild-type allele (in genome)
should in theory incorporate the foreign DNA segment of interest. Subsequently, the ES cells
must be screened using a negative selection method – antibiotics like neomycin – and
cultivate the cell culture. Genotyping via Southern Blotting can be carried out as a
confirmation procedure (Iwatsuki et al. 2010).
Generated Foxk2+/LacZ
ES cells are then injected into the host blastocyst which can then be
implanted into a female foster mouse or surrogate mother. The foster mouse will give birth to
chimeric mice. Chimeric mice are crossed with normal mice to obtain Foxk2+/LacZ
mice;
heterozygotes can then be crossed to generate homozygous Foxk2LacZ/LacZ
mice. The tip of the
tail is then cut off, and used for genotyping.
Analysis of Reporter gene expression is carried out via X-gal staining. In most cases, three
samples are strained: Foxk2+/LacZ
, Foxk2LacZ/LacZ
, and the wild-type (control). Histological
slices or sections of the organism can be photographed.
Fluorescent immunohistochemistry using antibodies against Foxk2, and β-galactosidase can
further be used to validate the results obtained from the X-gal staining of the three strains
(Foxk2+/LacZ
, Foxk2LacZ/LacZ
, and the wild-type) (Iwatsuki et al. 2010).
2. Figure 5: Target vector construct (LacZ = gene for production of β-galactosidase; Neo =
Neomycin (negative selection method); HSV-tk = lethal marker (positive selection
marker); 5’Arm = region homologous to genomic DNA at 5’ end; 3’Arm = region
homologous to genomic DNA at 3’ end)
3. REFERENCES
Iwatsuki, Ken, Masatoshi Nomura, Atsushi Shibata, Reiko Ichikawa, Patricio L.m. Enciso,
Lixiang Wang, Ryoichi Takayanagi, Kunio Torii, and Hisayuki Uneyama. "Generation and
Characterization of T1R2-LacZ Knock-in Mouse." Biochemical and Biophysical Research
Communications 402.3 (2010): 495-99. Web.