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What is a starter cell?

What is a starter cell?


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I am reading the paper Cooperative Subnetworks of Molecularly Similar Interneurons in Mouse Neocortex and a term "starter cell" apears there (page 6):

This yielded tissue sections where SOM or VIP starter cells carry both nucleus-localized GFP and cytoplasmic mCherry

Does anyone know what a starter cell is? (I couldn't find it on the web)


Nice question - this terminology isn't referring to a special type of cell or anything, but to a peculiarity of the technique they are using.

They are labeling a subset of cells with rabies virus; rabies then travels in retrograde fashion to presynaptic cells, and labels those as well.

The "starter cells" are those initially infected cells, where the tracing "starts." This reference more explicitly states this terminology, and talks about the technique extensively, I would strongly recommend it for helping to understand the paper you are reading.


Animals are a kingdom of living things. Learn about this incredible group of organisms.

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Our brilliantly simple book will take you through the fundamentals of biology in a way that is easy to follow and avoids difficult science jargon. Easy and enjoyable to read, the book introduces topics such as genetics, cells, evolution, basic biochemistry, the broad categories of organisms, plants, animals, and taxonomy.

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Animals are a kingdom of living things. Learn about this incredible group of organisms.

Plants are a kingdom of living things that are able to produce food using the sun’s energy.

Biology is a massive area of study. Learn about the different fields of biology.

Our brilliantly simple book will take you through the fundamentals of biology in a way that is easy to follow and avoids difficult science jargon. Easy and enjoyable to read, the book introduces topics such as genetics, cells, evolution, basic biochemistry, the broad categories of organisms, plants, animals, and taxonomy.

Also available from Amazon, Book Depository, and all good bookstores.

FREE 6-Week Course

Enter your details to get access to our FREE 6-week introduction to biology email course.

Learn about animals, plants, evolution, the tree of life, ecology, cells, genetics, fields of biology and more.

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EMBRACING UNCERTAINTY AND FEAR IS A POWERFUL STRATEGY FOR SUCCESS

As an experimental biologist, I learned quickly that although a scientific result may be predictable based on the hypothesis and the experiment designed to test it, unpredictable outcomes are common, and that some of the most interesting outcomes arise as a function of unpredictability. So, too, can a career benefit from unpredictability. Under my beach umbrella, I had no way of knowing that my future would someday be filled with a dizzying number of federal government experiences, without which I wouldn’t know today how the priorities for the U.S. research enterprise are determined, or how it is organized and funded. For example, when I first arrived in Washington, D.C., I learned that each year the OSTP and the Office of Management and Budget (OMB) release a memo for the federal agencies that fund science and technology (S&T) that describes the Administration’s priorities for the next federal budget, essentially a “road map” for where science funding will go 2 years from then.

I also didn’t know that I would become the primary author of a national policy document, the 2012 National Bioeconomy Blueprint (NBB Maxon, 2012), foreshadowed by the FY2012 OSTP and OMB S&T priorities memo (Orszag and Holdren, 2010) Writing a document meant to shape the future of U.S. biotech policy was intimidating. At the same time, I knew my science chops would help me define the core questions and come up with reasonable strategies. While developing the NBB, I was also responding to urgent happenings such as the Deepwater Horizon Oil Spill, the Fukushima earthquake and tsunami, and a number of other urgent OSTP activities (Office of the Press Secretary, The White House, 2016) that served to make every day an unpredictable and exciting one.

Eventually I came to embrace uncertainty and the fear of not being perfectly prepared for job duties with which I had little experience. I began to see them as helping me to develop skills of resilience and resourcefulness. Now, as the Associate Laboratory Director for Biosciences at Lawrence Berkeley National Laboratory, I see these as my main valuable assets when multiple competing priorities and intimidating assignments present themselves, as they always do. For example, when I first testified before Congress (Figure 1) in 2015 (Maxon, 2015), I did my best to overcome my fears by imagining the experience might be similar to my grad school oral exam. (Perhaps my grad school experience prepared me for more than I realized when I began this essay!) My past experience has had other ways of leading to interesting professional opportunities: to this day, my previous work on the NBB continues to result in international invitations for me to describe the development and outcomes of our national strategy.

FIGURE 1: Mary Maxon, Associate Laboratory Director for Biosciences at Lawrence Berkeley National Laboratory, has testified twice to the U.S. House of Representatives Committee on Science, Space, and Technology: here on March 14, 2018, in a session on world-leading innovations in science from the Department of Energy’s national labs. Photo credit: House Committee on Science, Space, and Technology—Majority.


The finishing kick and critical size

It has been known for many years that slowly growing cells have a long G1, and only when these cells have grown to 'critical size' can they pass through Start. In the finishing kick hypothesis, critical size is equivalent to stored carbohydrate that is, the hypothesis predicts that the size-correlated parameter being measured by the cell is glycogen plus trehalose. When enough carbohydrate is stored for successful passage through this energy- and material-intensive part of the cell cycle, this is somehow sensed (perhaps via some glucose-related metabolite such as glucose 6-phosphate and the cyclic AMP pathway), a signal is sent (again, perhaps via the cyclic AMP pathway), the carbohydrate is liquidated, ATP is produced, and a burst of metabolism, nucleotide synthesis, protein synthesis and all the other events of Start ensue. The late-G1 peak in expression of ribosome and protein synthesis genes noted by both Klevecz et al. [6] and Tu et al. [7] is explained by the need for a burst of protein synthesis. Interestingly, most of the mutations affecting critical cell size either affect the synthesis of G1 cyclins (for example, CLN3-1, whi3 and whi5) or are related to (though not actually in) the cyclic AMP pathway (sch9 and sfp1) [24]. Oscillations in other metabolites and sets of genes would be explained as downstream effects of the oscillation in stored carbohydrate and of the metabolic burst.

The finishing-kick hypothesis can only explain critical size and Start in slowly growing cells. Cells growing rapidly on abundant glucose have little or no stored carbohydrate, and in any case no need for a metabolic burst, and the finishing-kick hypothesis is irrelevant to such cells. But there is also evidence that cells use multiple mechanisms for controlling the time of Start [23], and the mechanisms that apply in fast-growing cells may be quite different from those in slow-growing cells [25].

It is clear that glucose-limited yeast do undergo a metabolic oscillation superimposed on their cell-cycle oscillation. Whether this metabolic oscillation is primarily for temporal compartmentalizing of different metabolic process, or primarily for managing Start under difficult circumstances, or whether both hypotheses are true, remains to be seen. One promising avenue for distinguishing the hypotheses is the study of mutants that do not store any glycogen or trehalose [2, 26]. Such mutants are alive, but with aberrant cell cycles. At present, phenotypic analysis is not detailed enough to distinguish the two hypotheses, but in principle, mutants that lack storage carbohydrate should allow some interesting experiments.


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3 Main Stages for Translation of RNA | Cell Biology

The following points highlight the three main stages for translation of RNA and protein synthesis. The stages are: 1. Initiation of Polypeptide 2. Elongation of Polypeptide: 3. Termination of Polypeptide.

Translation of RNA: Stage # 1. Initiation of Polypeptide:

Ribosomes exist as separate large and small subunits. The first step in translation involves the binding of the small ribosomal subunit to the mRNA. Translation usually begins at the sequence AUG, sometimes GUG, which encodes methionine and is known as the trans­lation initiation codon.

The small subunit binds to the mRNA at a specific point (Shine-Dalgarno sequence) upstream of the AUG. In eukaryotes, the small ribosomal subunit recognizes the cap structure at the 5′ end of the mRNA. It then moves downstream until it encounters the first AUG. A tRNA charged with methionine binds to the AUG located by the small ribosomal sub-unit.

Initiation of polypeptide chain occurs with the involvement of initiation factors, ribosomal subunits and amino acyl-tRNA complex.

A number of accessory proteins called initiation factors are required for initiation. Bacteria have three, known as IF1, IF2, and IF3. Initiation begins with binding of IF1 and IF3 to the small ribosomal subunit. This helps to prevent binding of a large subunit before the mRNA has bound. Next, IF2 complexed with GTP binds the small subunit. Its purpose is to assist binding of the initiator tRNA.

The small subunit then binds the mRNA and locates the AUG initiation codon. The initiator tRNA charged with methionine binds to the complex and IF3 is released. Then large ribosomal subunit binds to the initiation complex to from a com­plete functional ribosome. This is accompanied by the release of IF1 and IF2 and hydrolysis of GTP (Fig. 16.10).

Formation of Formyl-methionyl tRNAƒmet:

In prokaryotes,. methionine carries a formyl group (-CHO) and hence it is called N-formyl methionine. The’ initiation of synthesis takes place through initiation tRNA. It is abbreviated as tRNAƒmet. The initiation tRNA or tRNAƒmet forms a complex with methionine called methionyl tRNAƒmet.

The amino group of methionine is blocked by formyl group to form N formyl methionyl tRNAƒmet. This reaction is catalysed by a transformylase.

Binding of Initiation factors with 305:

Small ribosomal subunit binds IF1, IF3. Next, IF2 com­plexed with GTP binds the small subunit.

Binding of 30S with mRNA:

Ribosomes are the sites of protein synthesis and are found disso­ciated into subunits (505 & 305). The 305 subunit attached at the AUG codon of the mRNA form­ing the mRNA-30S complex. The process requires the initiation factor IF3.

Binding of the fmet-tRNAfmet with 305- mRNA Complex:

The fmet-tRNAfmet attaches with the 30S-mRNA complex to give rise to the initiation complex 305-mRNA-fmet-tRNA/met. This reaction is facilitated with initiation factor IF2. IF3 is released in this step.

Association of Ribosomal Subunits:

The ini­tiation complex formed then associates with SOS subunit to reconstitute 70S ribosome. During this process OTP is converted to GDP and the initia­tion factor IF1 and IF2 are released.

There are minor differences in the initiation of polypeptide chains In between prokaryotes and eukaryotes.

In eukaryotes there are more Initiation factors. These factors are elF1, elF2, elF3, elF4A, elF4B, elF4C, elF4D, elF4F, elF5, elF6. The elF2, elF3, elF4A and elF4F con­tain multiple polypeptide chains, but others are simple polypeptides elF2 and elF3 are analo­gous to IF2 and IF3 of prokaryotes.

Formation of Ternary Complex:

OTP binds to elF2 which increases its affinity for met- tRNAmet. This met-tRNAmet associates with elF2 – GTP complex forming a ternary complex, i.e., met-tRNAmet-elF2-GTP. The initiator tRNA is not formylated in eukaryotes.

Association of Ternary Complex with 40S Subunit:

The ternary complex associates with 40S subunit, to form 43S initiation complex. The factor elF2 has three subunits, namely α, β, γ. The elF2α binds to GTP, elF2γ binds to met-tRNA and elF2βmay be a recycling factor.

Binding of mRNA with 43S Initiation Com­plex:

The mRNA at its 5′ end binds with 43S ini­tiation complex. This reaction depends on elF3 and the binding of mRNA is assisted by elF4F, elF4B and a high energy bond of ATP.

Association with 60S Subunit:

After associa­tion of the S’ end of mRNA, the initiation com­plex moves towards 3′ end in search of initiation codon AUG and then also associates with 60S subunit.

This association of 60S subunit with the initiation complex requires the factor elFS, because it helps in releasing elF2 and elF3. elF2 is released as a binary complex, elF2-GDR The 40S-60S joining reaction really depends on elF4C, and the GTP of the initiation complex is hydrolysed, when SOS ribosome is reconstituted.

Translation of RNA: Stage # 2. Elongation of Polypeptide:

Elongation of polypeptide has been rep­resented in Fig. 16.11.

A number of accessory proteins are required for elongation. In proka­ryotes, two elongation factors, EF-Tu and Ef-Ts, are involved. EF-Tu is associated with entry of a tRNA into the A site of ribosome. Ef-Tu binds charged tRNAs in association with GTP. Following entry to the A site, the GTP is hydrolyzed and EF-Tu is released bound to guanosine diphosphate (GDP).

Before another tRNA can bind, EF-Tu must be regenerated with the help of Ef-Ts.

First, EF-Ts displaces GDP by binding to EF-Tu a new molecule of GTP then replaces EF-Ts (Fig. 16.12). In eukaryotes, a com­plex protein called eEF-1 brings the tRNA to the A site. Again, the reaction is associated with hydrolysis of GTP.

Binding of AA-tRNA at A Site of Ribosome:

The larger subunit of ribosome has two sites where two molecules of tRNA are attached. These are called P site (peptide site) and A site (aminoacyl site). fmet-tRNAƒmet at first comes to the A site, then to the P site to make the A site available for the next aminoacyl tRNA. For the attachment of AA-tRNA to A site, a molecule of GTP and elongation factors Ef-Tu and EF-Ts are required.

Ef-Tu forms a ternary complex with AA-tRNA and GTP (AA-tRNA-GTP- EF-Tu). The formation of this complex is catalysed by EF-Ts. These complex deposits AA-tRNA at A site and GDP, EF-Tu are released along with the phos­phate group.

Formation of Peptide Bond:

This is a cata­lytic reaction during which a peptide bond is formed between the free carboxyl group (-COOH) of the peptidyl tRNA at the P site and the free amino (-NH2) group present with amino acyl tRNA at the A site.

The enzyme involved in this reaction is peptidyl transferase and is an internal part of SOS ribosomal subunit. After the formation of peptide bond, the tRNA at P site is de-acylated and the tRNA at A site carries the polypeptides.

Translocation of Peptidyl tRNA from A to P Site:

The peptidyl tRNA present at A site is then trans-located to P site.

For translocation of peptidyl tRNA from A site to P site, there are two models:

(i) According to two sites (A, P) model, de-acylated tRNA is liberated from P site, and with the help of one GTP molecule and an elongation factor EF-G, the pep­tidyl tRNA is trans-located from A to P site. The elongation factor EF-G, binds to ribosome, is released on hydrolysis of GTP.

(ii) According to three sites (A, P, E) model, initially the aminoacyl end of the t-RNA bound to the A site moves to the P site on SOS subunit at the time of peptide trans­fer, but only later during translocation, the anticodon end of this tRNA moves from A to P site on 30S subunit. This latter step requires action of elongation factor EF-G.

In this model, thus, there is an intermediate state, when anticodon end of this tRNA is still on A site (on SOS sub- unit), while the aminoacyl end occupies P site (on SOS subunit). A third tRNA binding site, E was also recognized on SOS subunit, through which tRNA leaves the ribosomes.

Translation of RNA: Stage # 3. Termination of Polypeptide:

Termination of the polypeptide chain is brought about by the presence of any one of the three termination codons, namely UAA, UAG and UGA.

In prokaryotes, termination codons are recognized by one of the two release factors, RFl and RF2. Of these release factors, RF1 recognizes UAA and UAG and RF2 recog­nizes UGA. They help the ribosome to recog­nize these triplets. A third release factor RF3 seems to stimulate the action of RF1 and RF2. In eukaryotes, there is only one release factor (eRFI).

Release of Polypeptide:

For release action, the poly-peptidyl tRNA must be present on P site and release factors help in splitting of the carboxyl groups between the polypeptide and the last tRNA carrying this chain (Fig. 16.13). Polypeptide is thus released and the ribosome dissociates into 2 subunits with the help of IF3.

In eukaryotes, there is only one release factor – eRFI. GTP seems to be necessary for binding of this factor to ribosome. GTP is deducted after the termination step has occurred which may be a prerequisite for the release of RF1 from the ribo­some.

Modification of the Released Polypeptide:

The released polypeptides are modified in vari­ous ways. An enzyme deformylase removes the formyl group of first amino acid methionine. Due to the action of certain other enzymes, exo-amino-peptidases, amino acids may be removed a tertiary structure. In this manner, these proteins from either the N-terminal end or the C-terminal ultimately become functional enzymes, end or both.

The polypeptide chain singly or in association with other chain also folds to take up a tertiary structure. In this manner, these protein ultimately become functional enzymes. The whole process of translation is present association with other chain also folds to take up ted in Fig. 16.14.


Culturing Jurkat T-cells - (Nov/30/2007 )

for some reasons we plan to start with Jurkat T-cells but have no experience I need some basic information:

adherent or not?
are there sublines, and which is commonly used?
generation time?
difficulty of handling?
other special information?

Non-adherent, there are sublines that I know of but those are transfected ones.
Generation time is around 24 hours if I'm not mistaken and they are easy to handle.

What do you need to do with them that you can't do with other cells?

Non-adherent, there are sublines that I know of but those are transfected ones.
Generation time is around 24 hours if I'm not mistaken and they are easy to handle.

What do you need to do with them that you can't do with other cells?

thanks vairus we are interested in PKCtheta which is known to be expressed in T-cells PKCtheta is raely expressed in most other types of cells

still another question: are Jurkat cells oncogenic or just non-oncogenic immortal?

for some reasons we plan to start with Jurkat T-cells but have no experience I need some basic information:

adherent or not?
are there sublines, and which is commonly used?
generation time?
difficulty of handling?
other special information?

Our group have been growing Jurkats for many years. They are very easy to maintain if conditions are kept constant i.e.

Use the best quality FBS/FCS and BATCH TEST IT.
Start the cells in Tissue Culture flasks, for example a T75.
Never "over split" the cells i.e. 1:10- 1:20 split ratio. BUT AT FIRST when they are recovering from cryopreservation, go for 1:2/1:4 splits.
Once they are established, grow the cells in Techne Stirrer Bottles. this allow you to grow 100's of millions of cells for your experiments.

Our groups reference papers:

PNAS Beltan et al Dec. 19 2000, Vol 97, No.26, pp 14602-14607

PNAS Beltran et al June 25 2002, Vol 99, No.13, pp 8892-8897

thank you Rhombus, very helpful information I will start as you advice

Are these cells different than the Jurkat cells? Just curious because Jurkat cells too have the same properties described as above.

I am growing Jurkat cells for the first time too and although I think things are going well, I just wanted to check on a few little things:

1) The RPMI media that I am using appears to be slightly orange now that I have refrigerated it in 4º, not the pink color it was when I received it. I know that this is an indication of a PH change, is this normal and okay?

2) I am growing the cells in 75cm2 vented flasks and, aside from the reccomended volume limitations on this particular flask, is there an optimum volume that is best for maintaining these cells?

3) I am maintaining them at a density between 1 and 2 x 10(6), this is more dense that is reccomended but they seem to be doing okay, is there any problem with this?

4) When I am preparing to mix the FBS with the media, can I put the FBS in a 37º water bath to defrost? Also, I am adding Pen/Strep antibiotic-can I defrost this in 37º waterbath?

I am growing Jurkat cells for the first time too and although I think things are going well, I just wanted to check on a few little things:

1) The RPMI media that I am using appears to be slightly orange now that I have refrigerated it in 4º, not the pink color it was when I received it. I know that this is an indication of a PH change, is this normal and okay?

2) I am growing the cells in 75cm2 vented flasks and, aside from the reccomended volume limitations on this particular flask, is there an optimum volume that is best for maintaining these cells?

3) I am maintaining them at a density between 1 and 2 x 10(6), this is more dense that is reccomended but they seem to be doing okay, is there any problem with this?

4) When I am preparing to mix the FBS with the media, can I put the FBS in a 37º water bath to defrost? Also, I am adding Pen/Strep antibiotic-can I defrost this in 37º waterbath?

In our experience it is best to keep the cells at between 250,000 and 750,000 cells/ml. WE DO NOT USE ANTIBIOTICS. it masks POOR TECHNIQUE. And a s stated previously, the cells are best grown in TECHNE stirrer bottles. this keeps them in suspension and more importantly STOPS THEM AGGREGATING.

If you look after them properly they are one of the easiest cells to grow in large numbers.


The human body comprises more than 200 types of cells, and every one of these cell types arises from the zygote, the single cell that forms when an egg is fertilized by a sperm. Within a few days, that single cell divides over and over again until it forms a blastocyst, a hollow ball of 150 to 200 cells that give rise to every single cell type a human body needs to survive, including the umbilical cord and the placenta that nourishes the developing fetus.

Basic cell biology

Each cell type has its own size and structure appropriate for its job. Skin cells, for example, are small and compact, while nerve cells that enable you to wiggle your toes have long, branching nerve fibers called axons that conduct electrical impulses.

Cells with similar functionality form tissues, and tissues organize to form organs. Each cell has its own job within the tissue in which it is found, and all of the cells in a tissue and organ work together to make sure the organ functions properly.

Regardless of their size or structure, all human cells start with these things in common:

  • A nucleus that contains DNA, the genetic library for the entire body. Different cells read and carry out different instructions from the DNA, depending on what those cells are designed to do. Your DNA determines virtually everything about your body, from the color of your eyes to your blood type and even how susceptible you are to certain diseases. Some diseases and conditions, such as color blindness, also are passed down through DNA.
  • Cytoplasm – the liquid outside the nucleus. The cytoplasm contains various components that make the materials that the cell needs to do its job.
  • The cell membrane – the surface of the cell, a complex structure that sends
    and receives signals from other cells and lets material in and out of the cell. Cells have to be able to communicate to work together in tissues and organs.

Most cells divide. Shortly before division, the DNA replicates and then the cell divides into twodaughtercells. Each has a complete copy of the original cell’s DNA, cytoplasm and cell membrane.

About stem cells

Stem cells are the foundation of development in plants, animals and humans. In humans, there are many different types of stem cells that come from different places in the body or are formed at different times in our lives. These include embryonic stem cells that exist only at the earliest stages of development and various types of tissue-specific (or adult) stem cells that appear during fetal development and remain in our bodies throughout life.
Stem cells are defined by two characteristics:

  • They can make copies of themselves, or self-renew
  • They can differentiate, or develop, into more specialized cells

Beyond these two things, though, stem cells differ a great deal in their behaviors and capabilities.

Embryonic stem cells are pluripotent, meaning they can generate all of the body’s cell types but cannot generate support structures like the placenta and umbilical cord.

Other cells are multipotent, meaning they can generate a few different cell types, generally in a specific tissue or organ.

As the body develops and ages, the number and type of stem cells changes. Totipotent cells are no longer present after dividing into the cells that generate the placenta and umbilical cord. Pluripotent cells give rise to the specialized cells that make up the body’s organs and tissues. The stem cells that stay in your body throughout your life are tissue-specific, and there is evidence that these cells change as you age, too – your skin stem cells at age 20 won’t be exactly the same as your skin stem cells at age 80.


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Comments:

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  6. Yozshulkis

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