Week 2: Gene Expression Analysis and Mouse Breeding

Hi everyone! Welcome back to my Week 2 project update 🤩 Also side note: I finally have gained a skill I should have had all along – navigating public transport. The commute to UCSF is a ride, but now I can safely say I know what I’m doing. Yay!

Gene Expression Analysis and Cell Typist Annotations

Carrying forward from last week, I have been using CellTypist to create automated and manual cell type annotations on my scRNA-seq data. CellTypist allows me to train and test a model on a reference dataset of multiple primitive cell types to make predictions on the specific kinds of cells in the query/target dataset. 

I have also been using pseudo-bulk-based differential expression analysis through the pyDESeq2 package, which essentially aggregates the gene expression of the cells in the dataset. This allows the analysis to be more robust to dropouts, or cases where the genes are expressed differently by a cell. Essentially, if the program is trained to see only one kind of gene expression due to using a small sample size (for instance, treating each cell as its own sample), it won’t pick up on the same gene being expressed in a different way. For this reason, we choose to create bigger samples (grouping all the cells into one big sample), so the program can see all the different ways a gene is being expressed! 

So what did I analyze with pyDESeq2? I used this program to see differences in gene expression between migrating PGCs at different timepoints — specifically, at E9.5, E10.5, and E11.5 (timepoints in embryonic development). Next week, I will investigate the differences in gene expression between anterior and posterior migrating PGCs at the first two timepoints (as by E11.5, the PGCs have mostly reached the gonad already).

Wet-Lab Update

My advisor and I genotyped a mouse litter to identify mice to be used in future experiments. We took tiny samples of their tails to get DNA and then performed PCR on those samples, followed by gel electrophoresis. 

But first…

What is genotyping?

Genotyping helps us determine…you guessed it…the genotype of the mice! We are looking for mice with very specific genetics, so we want to check that they are homozygous or heterozygous for the genes of interest. After plug checking the mice in the morning, getting tail samples (which we will dissociate to get DNA) from the mice that were to be genotyped, and attending the Wagner Lab meeting, I prepared the samples for the PCR. Polymerase Chain Reaction (PCR) is a DNA amplification technique, and DNA amplification is important because for the best readout, we want as many DNA fragments as possible. PCR involves 3 main steps: denaturation, annealing, and extension. During denaturation, the DNA strands are separated due to high temperatures, producing two single strands. In annealing, cooler temperatures allow primers (a very short stand of DNA that serves as a starting point for DNA synthesis) to bind to the complementary parts of the DNA. As a result, primers are highly specific and targeted, so we add primers that will specifically bind to the part of the DNA we want to amplify (the part of the DNA that contains the alleles for the gene of interest). In extension, DNA polymerase uses the primers as a guide to finish making the complementary sequence to make small double-stranded DNA again. Now, PCR repeats the cycle 25-40 times, allowing amplification again and again. 

After running the PCR on my samples, I performed gel electrophoresis, which allows DNA fragments to separate based on size and charge. DNA is negatively charged (a property derived from the negative charges on the oxygen in the phosphate group) — the gel has a positive and negative end, so once the gel runs, the DNA moves to the positive end. When making the agarose gel, we use “combs” to create dents, known as “wells” in the gel where we can load our samples. Once the gel is loaded with the samples, we “run” it by applying an electric current. As the current is applied, the smaller and more negatively charged DNA fragments move further to the positive end.

Here is a picture of a gel I ran that was only for the genotyping of 3 genes:

Since different alleles of a gene are different sizes, we can tell whether a mouse is homozygous for either of the genetic alleles or heterozygous for a gene based on the sizes of DNA fragments we observe. In the readout for the gel, this can be seen by “bands” at different vertical positions across the gel — with bands closer to the positive end having a smaller number of base pairs and the bands farther having a greater number of base pairs. If we see two bands, we know there must be DNA fragments of different sizes, so the mouse is heterozygous for that gene. If we see one band, we know it is homozygous, and we have to check the relative position of the band — aka the relative size of the DNA fragments — to check whether it is the wild type or mutant allele. This we do using a DNA ladder, which will tell us where fragments of certain base pair lengths, like 100, 200, 300, etc. base pairs, should land in the gel. 

In this genotyping sample, you can only see one band amplified for each gene. That means all the mice were homozygous for all the genes!

The results of the gel were used to identify mice that were homozygous at 3 gene loci: at the CRISPR-associated protein 9 (Cas9), reverse tetracycline transactivator (rtTA), and octamer-binding transcription factor 4—green fluorescent protein (Oct4GFP) loci. Why do we care about Cas9, rtTA, and Oct4GFP? These 3 proteins combined are important for genomic editing (handy when we want to edit into each cell a barcode that will label it so we can track it) and the regulation/activation of gene marker expression that enables us to visualize germ cell migration and germ cell fate. 

Remember FACS? FACS is able to separate the germ cells from the somatic cells because the germ cells express Oct4GFP, and the somatic cells don’t!

In addition to the above, I went with my advisor to attend a very enlightening talk on the Neuropsychiatry of Lupus by Dr. Betty Diamond from the Feinstein Institutes for Medical Research!