I used pet16b vector to clone my gene of interest to further purify the protein .I added the fluorescent tag at the c-terminal for further microscopy. After cloning ,samples were sent to sequence for verification . When it comes to protein expression, after cells harvest protein doesn't express and fluorescent tag lost. what may be the problem?
Hello, when you say that you sent the expression vector for sequencing, was the the results positive? It is important that your fluorescent protein sequence is "in frame".
If you are doing transfection, you should have a control that contains the fluorescent protein (GFP or RFP) alone, without the target protein. If you don't even have this control working, it means some procedure has gone wrong.
(1)Not following the stepwise process that leads to the formation of all lineages of blood precisely haemotopoiesis.
(2)injection of exogenous DNA into the Genomes.
When there is a viral infection of the egg or injection into a mouse at the embroyonic stem cells into a blastocytes.
(3)Not using a site method specific recombination in the knock -in approach ,using a random insertion ,it can lead to loss ,although the recent example of knock-in is transgenic mouse is the Mirror 144/451 GEP line,in which GEP is expressed in adult and fetal liver erythroid cells.A concern for the knock-in approach is that it may result in haploinsufficiency, infleucing not only expression of the FP reporter but also the phenotype of resulting in animal loss of one Runx 1 allele affected the distribution of Hscs in the embryo .this problem was overcome by linking the sequences gene through an internal entry site (lRES) to create a dicistronic fusion MRNA.
(4)Bacterial artificial chromosmes (BAC) accomodate large DNA sequences allowing cloning of all or most of the endogenous regulatory elements required to recapisulate the normal pattern of gene expression such as those encoding a FP.
(4)Not using the invaluable tools for the studying the development of the haematopoietic system examples using transgenic mouse line expressing flouresent proteins.
(5)when there is stimulated by light of specific wavelengths.
(6)the availability of flouresent variants allows the researcher to select combinations that minimise spectral overlap.examples the combinations of GEP/CEP and GEP /YEP exhibit significant emission overlap.
(7)the choice if planing a genetic background is always considered.
(8)Analysing of flouresent protein expression using microscopy is also considered.
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