Can someone explain this molecular biology to me?

[BilZ0r]

So I see this abstract:
http://www.ncbi.nlm.nih.gov/entrez/...&dopt=Abstract&list_uids=16014726&query_hl=10

And I'm like "Wow, getting cells that express the D1 receptor to express a bung NR1 subunit, thats a good trick, how in the name of fuck do you do that?"

So the methods say:

The targeting construct contained 7.0 kb of 129/Sv-derived Drd1a receptor genomic sequence. The NcoI site that contained the initiation codon of the Drd1a locus was converted to a BglII site, effectively destroying the initiation codon. The NR1 cDNA (gift from Gerald Rameau, New York University, New York, NY), containing an open reading frame with a hemagglutinin (HA)-epitope tag at a HincII site 27 amino acids from the N terminus, the N616R mutation, and a polyadenylation sequence, was cloned between this BglII site and a downstream HindIII site, replacing ~130 bp of the endogenous Drd1a gene. The resulting targeting construct had ~3 kb of genomic Drd1a DNA 5' and ~4 kb of Drd1a DNA 3' to the mutant NR1 cDNA. A simian virus 40 -neomycin resistance (Neo) cassette that is flanked by loxP sites was inserted immediately after the NR1 cDNA to allow selection in embryonic stem cells.

The linearized targeting vector was electroporated into V6.4 embryonic stem cells derived from a cross of 129/Sv and C57BL/6 mice. G418-resistant clones were expanded and screened by Southern blotting. For Southern blots, genomic DNA was digested with HindIII and probed with a 300 bp fragment of DNA from the Drd1a receptor locus immediately 5' to the targeting vector (see Fig. 1 A). Homologous recombination resulted in a product that was ~4 kb larger than the wild-type (WT) product. Of 360 clones screened, six were correctly targeted, and one gave germline transmission. Progeny carrying the mutant allele were genotyped using PCR primers to detect the presence or absence of the neomycin resistance gene (5'-GCTATTGGGCGAAGTGCCGG-3' and 5'-GGCAAGCAGGCATCGCCATG-3'). Southern blot on genomic DNA from Neo-positive pups confirmed the PCR results. Additional genotyping was performed using primers flanking the inserted sequence (5'-GACCAGGAAGAGGCCCCAGACT-3' and 5'-GTCACCTTGGACCGCAGGTGTC-3') and a primer from the NR1 cDNA (5'-CATGCTTGCGCGTGCTCAGCAC-3'). The mice containing the targeted mutation Drd1a Grin1-N616R (designated D1R-NR1m mice) were maintained on a mixed C57BL/6 x 129/Sv background. Control animals were littermates of D1R-NR1m mice that did not contain the targeted mutation (designated WT mice).

And I don't understand how that led to D1 receptor expressing cells exclusively expressing a shit NR1 subunit? Or is the idea that when D1 receptors are expressed, the mRNA gets processed into two proteins, the D1 and the NR1 polypeptide, so in D1 expressing cells, some NR1 is the bung version?

 

[ek-stase]

AFAIK, the targeting vector with the DNA for the shit subunit was inserted into the cell and was incorporated into the daughter cells' DNA by homologous recombination during mitosis, effectively replacing a specific part of the DNA (the part that codes for the good subunit). It does this by sticking around the DNA you want to replace and getting copied during DNA replication into the chromosome. Exactly how and why techniques like this work may or may not be well figured out, (the practice of Mol Bio can be more art than science at times as many techniques are used that aren't completely understood), in any case I don't know nor really care. But that is basically what happens if I understood that correctly. So in the daughter cells all the D1 receptors will have the bad subunit because the DNA for the good one has been (in whole or part) replaced. This is of course only in the daughter cells that have the vector incorporated, and these are selected by an antibiotic that kills those without the insert (thats why they added dna to the insert to confer resistance). Let me know if that helps
-ek

 

[mitogen]

So essentially the targeting vector targeted the right sequence such that the D1 gene was actually replaced by a sequence for the bunk NR1 subunit?

[PING] Bilz0r, hows the studies going?
I'm going to be doing some protein engineering next year with a guy named Peter Metcalf.

 

[BilZ0r]

^ I didn't think so... becasue then the D1 expressing cells wouldn't express D1 anymore... the idea is that somehow, only in d1 expressing cells, the NR1 subunit gets replaced by a bung version.

Studies are going good... ish... Giving a lecture in a couple of days to some 3rd years..

Peter Metcalf, the name rings a bell.

 

[ek-stase]

The whole D1 gene is not replaced, just the NR1 subunit part. The replacement section also confers antibiotic resistance which allows you to select those stem cells with the insert. You ten take those stem cells with your insert and make a mouse which will express the bad subunit in its D1 expressing cells.
-ek

 

[sorceror]

Peter Metcalf, the name rings a bell.

he was a lecturer of mine last year

university of auckland