Reprogramming Fibroblasts to iPS Cells Using mRNA

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Questions and Answers

What type of RNA is used to follow the kinetics and stability of the intracellular expressed protein?

rRNA

snRNA

mRNA (correct)

tRNA

Which of the following was NOT used to express transcription factors in the human fibroblasts?

T7 RNA polymerase

CRISPR-Cas9 system (correct)

IRES sequence

pTMA plasmid

How was the GFP coding region replaced in the pTMA plasmid?

Homologous recombination

Reverse transcription

Restriction enzyme digestion and ligation (correct)

Transposon insertion

What is the role of the IRES sequence in the pTMA plasmid?

Directs translation initiation (C) Signup and view all the answers

When did the expression of GFP reach its maximum level after a single transfection?

24 hours (A) Signup and view all the answers

What was the primary method used to examine the localization of the expressed transcription factors?

Fluorescence microscopy (A) Signup and view all the answers

What was the primary observation regarding the localization of the expressed transcription factors compared to GFP?

Transcription factors localized exclusively to the nucleus while GFP stayed in the cytoplasm (B) Signup and view all the answers

Based on the provided information, which of the following is NOT a transcription factor used in this study?

Pax6 (C) Signup and view all the answers

What is the purpose of using T7 RNA polymerase in the preparation of human iPS from foreskin fibroblasts?

To transcribe DNA into mRNA. (D) Signup and view all the answers

Why are RNA-induced pluripotent stem cells (RiPS) considered an improvement over traditional iPS generated using DNA vectors?

RiPS cells are less likely to integrate into the host genome, reducing the risk of malignancy or genetic defects. (B) Signup and view all the answers

Which of the following techniques is used to examine the pluripotency of the iPS colonies?

Anti-Nanog staining. (A) Signup and view all the answers

What is the approximate magnification used for visualizing the AP staining?

100x (D) Signup and view all the answers

Which of the following factors is NOT mentioned as being involved in the reprogramming process?

Valproic acid (A) Signup and view all the answers

What is the typical timeframe for peak expression of the proteins encoded by the RNA used for reprogramming?

24 hours post-transfection (D) Signup and view all the answers

What is the primary advantage of using a plasmid platform for the production of mRNA?

Production of capped and polyadenylated RNA, improving stability and protein expression. (B) Signup and view all the answers

What is the main motivation for using RNA-induced pluripotent stem cells (RiPS) instead of traditional iPS generated from DNA vectors?

RNA methods eliminate the risk of genome integration and potential genetic defects associated with DNA vectors. (B) Signup and view all the answers

What is the likely explanation for the persistent expression of transcription factors 7 days after the fifth transfection?

Endogenous genes of these factors are activated. (B) Signup and view all the answers

What evidence supports the idea that the expression of SSEA4 is due to activation of endogenous genes rather than transfection?

SSEA4 was not included in the transfected RNA. (B) Signup and view all the answers

What is the significance of the observed expression of Nanog 20 days after transfection?

It indicates the successful reprogramming of hFF cells into iPS cells. (C) Signup and view all the answers

What is the primary method used to visualize and analyze iPS colonies?

AP staining (C) Signup and view all the answers

What does the observation of primary iPS colonies 10 days after the fifth transfection suggest?

Reprogramming is a slow and gradual process. (A) Signup and view all the answers

What is the approximate number of iPS colonies obtained per $10^5$ hFF cells?

50 (B) Signup and view all the answers

What is the purpose of using DAPI staining in the experiment?

To visualize the nucleus of the cells. (C) Signup and view all the answers

What is the significance of the observation of negative MEF background in Figure 4H?

It suggests that the Nanog expression is not occurring in the MEF cells. (C) Signup and view all the answers

What is the role of irradiated murine embryonic fibroblasts (iMEFs) in the described procedure?

They act as a feeder layer for the hES cell culture. (B) Signup and view all the answers

What is the purpose of adding the antibiotic to the hFF medium after the 5th hour of incubation?

To prevent bacterial contamination. (B) Signup and view all the answers

Which of the following is not a component of the hES medium used to culture the hFF cells?

Fetal bovine serum (B) Signup and view all the answers

What is the role of the polyA sequence added to the pTMA plasmid?

It enhances the stability of the mRNA produced from the plasmid. (A) Signup and view all the answers

What is the significance of using a high-fidelity DNA polymerase in the PCR amplification of the TF cDNAs?

It minimizes the introduction of mutations into the amplified DNA. (B) Signup and view all the answers

What is the purpose of linearizing the pTMA plasmid with SalI before in vitro transcription (IVT)?

To create a template for the T7 RNA polymerase. (B) Signup and view all the answers

Why is the IVT process described as allowing "5' cap formation"?

It promotes the binding of the transcribed RNA to ribosomes. (C) Signup and view all the answers

What is the purpose of using conditioned medium after 10 days on iMEFs?

To provide additional nutrients and growth factors for the iPS cells. (B) Signup and view all the answers

Which of the following proteins were shown to be localized to the nucleus of hFF cells after transfection?

SOX2 (A), OCT4 (C) Signup and view all the answers

What is the purpose of the SalI restriction site in the context of the experiment described in the text?

To create a linear plasmid for transcription (A) Signup and view all the answers

Which of the following statements is true about the expression of the four factors (OCT4, SOX2, Nanog, and Lin28) in hFF cells?

The expression of all four factors decreases significantly after the fifth transfection. (A) Signup and view all the answers

Where is SSEA4 protein primarily localized in hFF cells after transfection?

Cell membrane (A) Signup and view all the answers

What is the main purpose of the experiments described in the text?

To determine the expression levels of specific proteins in hFF cells after transfection (A) Signup and view all the answers

What is the function of T7 RNA polymerase in the experiment?

To transcribe the DNA plasmid into RNA. (B) Signup and view all the answers

Which of the following experimental techniques was NOT used in this study?

Western blotting (C) Signup and view all the answers

What is the approximate percentage of hFF cells that continue to express the four factors (OCT4, SOX2, Nanog, and Lin28) after the fifth transfection?

25% (B) Signup and view all the answers

What is the purpose of the DNaseI treatment in the RNA isolation process?

To degrade any remaining genomic DNA contamination. (B) Signup and view all the answers

What is the primary function of the RNeasy columns used in the study?

To isolate and purify mRNA from a complex mixture of nucleic acids. (B) Signup and view all the answers

How is the RNA integrity confirmed before RT-PCR analysis?

Agarose gel electrophoresis of the RNA sample. (D) Signup and view all the answers

What is the purpose of using dT25 primers in the RT-PCR process?

To synthesize complementary DNA (cDNA) from RNA templates. (B) Signup and view all the answers

What is the significance of using a gene-specific primer in the RT-PCR process?

To ensure that only the desired mRNA transcript is amplified. (D) Signup and view all the answers

What is the purpose of including GFP in the RNA mixture for transfection?

To serve as an internal control for the transfection efficiency. (C) Signup and view all the answers

Why is the resulting cDNA mix five-fold diluted before being used as a template for PCR?

To reduce the concentration of cDNA and prevent PCR inhibition. (B) Signup and view all the answers

What is the purpose of the GAPDH primer set used in the PCR?

To normalize the expression levels of target genes. (B) Signup and view all the answers

Study Notes

Reprogramming Human Fibroblasts to Pluripotent Stem Cells Using mRNA

Induced pluripotent stem cells (iPS) are similar to embryonic stem cells (ESCs), capable of differentiating into any cell type and self-renewing.
Yamanaka's 2006 work showed successful iPS generation from somatic cells by transfecting DNA encoding four transcription factors (TFs): Oct4, Sox2, Klf4, and c-Myc.
Subsequent modifications, including using Lin28 and Nanog instead of Klf4 and Myc, and alternative DNA delivery methods (retroviral or lentiviral infection, plasmids, etc.), have been employed.
DNA integration into the genome poses risks of uncontrolled cell growth and potential malignancy due to unpredictable integration sites.

RNA Transfection for iPS Generation

A method using in vitro-produced mRNA encoding Oct4, Lin28, Sox2, and Nanog to reprogram human foreskin fibroblasts (hFFs) into iPS cells (RiPS) is presented.
This method avoids DNA integration.
Intracellular expression of these proteins was observed in at least 70% of cells in the first eight hours.
Continuous protein expression (supported by five consecutive transfections) was crucial for forming iPS colonies expressing alkaline phosphatase and other ESC markers.
This approach might replace DNA vectors for iPS generation.

Materials and Methods

hFFs were cultured and transfected with a mixture of mRNAs.
RNA integrity monitored through agarose gel electrophoresis and RT-PCR.
Detection of proteins through fluorescence and immunocytochemistry with specific antibodies (e.g., OCT4, SOX2)
Colonies, showing alkaline phosphatase activity, grew after five RNA transfections and differentiation onto iMEFs.

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Description

This quiz covers the process of reprogramming human fibroblasts into induced pluripotent stem cells (iPS) using mRNA technology. Learn about the key transcription factors involved, the advantages of RNA transfection over DNA integration, and the implications of this innovative method in stem cell research.